Modified glucosyltransferases for producing branched alpha-glucan polymers

ABSTRACT

Glucosyltransferase enzymes are disclosed herein that produce branched alpha-glucan polymer. Also disclosed, for example, are polynucleotides encoding these enzymes, as well as methods of producing branched alpha-glucan polymer.

This application claims the benefit of U.S. Provisional Application Nos.62/180,779 (filed Jun. 17, 2015) and 62/180,788 (filed Jun. 17, 2015),which are both incorporated herein by reference in their entirety.

FIELD OF INVENTION

The present disclosure is in the field of enzyme catalysis. For example,the disclosure pertains to the production of branched alpha-glucansusing modified glucosyltransferase enzymes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20160615_CL6480SequenceListing_ST25_ExtraLinesRemoved.txt created onJun. 14, 2016, and having a size of 740 kilobytes and is filedconcurrently with the specification. The sequence listing contained inthis ASCII-formatted document is part of the specification and is hereinincorporated by reference in its entirety.

BACKGROUND

Driven by a desire to find new structural polysaccharides usingenzymatic syntheses or genetic engineering of microorganisms or planthosts, researchers have discovered polysaccharides that arebiodegradable and can be made economically from renewably sourcedfeedstocks. One such polysaccharide is poly alpha-1,3-glucan, a glucanpolymer characterized by having alpha-1,3-glycosidic linkages. Thispolymer has been isolated by contacting an aqueous solution of sucrosewith a glucosyltransferase (GTF) enzyme isolated from Streptococcussalivarius (Simpson et al., Microbiology 141:1451-1460, 1995).

U.S. Pat. No. 7,000,000 disclosed the preparation of a polysaccharidefiber using an S. salivarius gtfJ enzyme. At least 50% of the hexoseunits within the polymer of this fiber were linked viaalpha-1,3-glycosidic linkages. S. salivarius gtfJ enzyme utilizessucrose as a substrate in a polymerization reaction producing polyalpha-1,3-glucan and fructose as end-products (Simpson et al., 1995).The disclosed polymer formed a liquid crystalline solution when it wasdissolved above a critical concentration in a solvent or in a mixturecomprising a solvent. Continuous, strong, cotton-like fibers wereobtained from this solution that could be spun and used in textileapplications.

While some advances have been made in producing linear glucan polymershaving a high percentage of alpha-1,3 glycosidic linkages suitable foruse in spinning fibers, it is believed that less attention has beendrawn to producing branched alpha-glucan polymers. To that end,disclosed herein are modified glucosyltransferases that can synthesizebranched alpha-glucan.

SUMMARY OF INVENTION

In one embodiment, the disclosure concerns a glucosyltransferase enzymecomprising a catalytic domain that comprises an amino acid sequence thatis at least 90% identical to amino acid positions 54-941 of SEQ IDNO:85, 54-927 of SEQ ID NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQ IDNO:91, 54-919 of SEQ ID NO:93, 54-905 of SEQ ID NO:95, or 54-889 of SEQID NO:97, wherein the catalytic domain lacks at least one motif selectedfrom the group consisting of:

-   -   (i) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:78,    -   (ii) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:79, and    -   (iii) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:80;        wherein the glucosyltransferase enzyme produces a branched        alpha-glucan polymer.

In another embodiment, the glucosyltransferase comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:85, SEQ ID NO:87,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97,and wherein the glucosyltransferase lacks at least one of motifs (i),(ii), or (iii).

Another embodiment concerns a polynucleotide comprising a nucleotidesequence encoding a glucosyltransferase enzyme as disclosed in the aboveembodiment, optionally wherein one or more regulatory sequences areoperably linked to the nucleotide sequence, and preferably wherein theone or more regulatory sequences include a promoter sequence.

Another embodiment concerns a method of preparing a polynucleotidesequence encoding a glucosyltransferase enzyme that produces a branchedalpha-glucan polymer. This method comprises:

(a) identifying a polynucleotide sequence encoding a parentglucosyltransferase enzyme that comprises a catalytic domain comprising:

-   -   (1) an amino acid sequence that is at least 90% identical to        amino acid positions 54-957 of SEQ ID NO:65, and    -   (2) the following three motifs:        -   (i) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:78,        -   (ii) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:79, and        -   (iii) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:80;        -   and            (b) modifying the polynucleotide sequence identified in            step (a) to delete and/or mutate at least one of motifs (i),            (ii), or (iii) encoded by the polynucleotide sequence,            thereby providing a polynucleotide sequence encoding a            glucosyltransferase enzyme that produces a branched            alpha-glucan polymer.

In another embodiment, the position of the amino acid sequence that isat least 90% identical to SEQ ID NO:78 aligns with amino acid positions231-243 of SEQ ID NO:65; the position of the amino acid sequence that isat least 90% identical to SEQ ID NO:79 aligns with amino acid positions396-425 of SEQ ID NO:65; and/or the position of the amino acid sequencethat is at least 90% identical to SEQ ID NO:80 aligns with amino acidpositions 549-567 of SEQ ID NO:65.

In another embodiment, the motif (i) comprises SEQ ID NO:78, motif (ii)comprises SEQ ID NO:79, and motif (iii) comprises SEQ ID NO:80.

In another embodiment, the parent glucosyltransferase enzyme cansynthesize poly alpha-1,3-glucan having at least 95% alpha-1,3glycosidic linkages and a weight average degree of polymerization(DP_(w)) of at least 100.

In another embodiment, modification step (b) comprises deleting at leastone of motifs (i), (ii), or (iii) encoded by the polynucleotide sequenceidentified in step (a).

In another embodiment, the glucosyltransferase enzyme of step (b)comprises a catalytic domain that does not comprise at least one aminoacid sequence that is at least 60% identical to SEQ ID NO:78, SEQ IDNO:79, or SEQ ID NO:80.

In another embodiment, the branched alpha-glucan polymer has anintrinsic viscosity and/or branching index that is reduced by at least30% compared to the intrinsic viscosity and/or branching index of polyalpha-1,3-glucan synthesized by the parent glucosyltransferase.

In another embodiment, the identifying step is performed (a) in silico,(b) with a method comprising a nucleic acid hybridization step, (c) witha method comprising a protein sequencing step, and/or (d) with a methodcomprising a protein binding step; and/or wherein said modifying step isperformed (e) in silico, followed by synthesis of the polynucleotidesequence encoding a glucosyltransferase enzyme that produces a branchedalpha-glucan polymer, or (f) using a physical copy of the polynucleotidesequence encoding the parent glucosyltransferase.

Another embodiment concerns a polynucleotide sequence produced accordingto the above embodiment, optionally wherein the polynucleotide sequencefurther comprises one or more regulatory sequences operably linked tothe polynucleotide sequence, preferably wherein the one or moreregulatory sequences include a promoter sequence. Another embodimentconcerns a glucosyltransferase enzyme encoded such a polynucleotidesequence.

Another embodiment concerns a reaction solution comprising water,sucrose, and a glucosyltransferase enzyme as disclosed herein.

Another embodiment concerns a method for producing a branchedalpha-glucan polymer. This method comprises (a) contacting at leastwater, sucrose, and a glucosyltransferase enzyme as presently disclosed,whereby branched alpha-glucan polymer is produced, and b) optionally,isolating the branched alpha-glucan polymer produced in step (a).

Another embodiment concerns a branched alpha-glucan polymer producedfrom any glucan synthesis method or reaction disclosed herein, or thatis a product of any glucosyltransferase enzyme disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

FIG. 1: Comparison of the main chain tertiary fold of Lactobacillusreuteri GTF (gray) and Streptococcus mutans GTF (black). The structureof the L. reuteri GTF includes a fifth domain (Domain V) that wastruncated from the structure of S. mutans GTF. The active site is alsoindicated and is formed by a cavity in the central domains (theso-called A and B domains); this location is based on spatial similaritywith similar domains in alpha amylases. The amino acid sequence of theS. mutans 3AIE GTF structure is SEQ ID NO:66, and the amino acidsequence of the L. reuteri 3KLK GTF structure is SEQ ID NO:67.

FIGS. 2A-O: Alignment of twenty-four GTF sequences with sequences ofportions of GTFs from S. mutans (3AIE, SEQ ID NO:66) and L. reuteri(3KLK, SEQ ID NO:67) for which crystallographic structures are known;single-letter amino acid code is used. GTF amino acid sequences thatproduced glucan with 100% alpha-1,3 linkages and high molecular weight(DP_(w) of at least 400 under the tested initial sucrose concentrations,see Table 4) are designated “++”. Those GTFs producing glucan with 100%alpha-1,3 linkages and a DP_(w) of at least 100 are designated “+−”.Other GTFs producing glucan with mixed linkages are designated “−−”.

FIG. 3: The sequence of the tested GTF enzymes in the vicinity of Motifs1a and 1b.The sequence region of Motifs 1a and 1b along with upstreamand downstream flanking reference sequence motifs are shown as boxedregions. Motifs 1a and 1b are located in box labeled “Insertion 1”. Thealignment in this figure represents a portion of the alignment in FIGS.2A-O.

FIGS. 4A and 4B: Visualization of Motif 1a through comparison of ahomology model of GTF 7527 (SEQ ID NO:65) based on the referencecrystallographic structures of S. mutans (3AIE, SEQ ID NO:66) (FIG. 4A)and L. reuteri (3KLK, SEQ ID NO:67) (FIG. 4B). The main chain folding ofthe homology model in each view is shown with dark lines while the mainchain folding of the reference structure is shown with lighter lines.The residues forming the catalytic sites in the referencecrystallographic structures are shown as Van der Waals spheres forreference. Motif 1a (between the arrows) is presented in both homologymodels as an open loop (black) extending into the solvent as aconsequence of there being no homologous segment to provide means toposition with respect to the remainder of the GTF catalytic domain.

FIG. 5: The sequence of the tested GTF enzymes in the vicinity of Motif2. The sequence region of Motif 2 along with upstream and downstreamflanking reference sequence motifs are shown as boxed regions. Motif 2is located in box labeled “Insertion 2”. The alignment in this figurerepresents a portion of the alignment in FIGS. 2A-O.

FIGS. 6A and 6B: Visualization of Motif 2 through comparison of ahomology model of GTF 7527 (SEQ ID NO:65) based on the referencecrystallographic structures of S. mutans (3AIE, SEQ ID NO:66) (FIG. 6A)and L. reuteri (3KLK, SEQ ID NO:67) (FIG. 6B). The main chain folding ofthe homology model in each view is shown with dark lines while the mainchain folding of the reference structure is shown with lighter lines.The residues forming the catalytic sites in the referencecrystallographic structures are shown as Van der Waals spheres forreference. Motif 2 (between the arrows) is presented in both homologymodels as an open loop (black) extending into the solvent as aconsequence of there being no homologous segment to provide means toposition with respect to the remainder of the GTF catalytic domain.

FIG. 7: The sequence of the tested GTF enzymes in the vicinity of Motifs3a and 3b. The sequence region of Motifs 3a and 3b along with upstreamand downstream flanking reference sequence motifs are shown as boxedregions. Motifs 3a and 3b are located in box labeled “Insertion 3”. Thealignment in this figure represents a portion of the alignment in FIGS.2A-O.

FIGS. 8A and 8B: Visualization of Motif 3a through comparison of ahomology model of GTF 7527 (SEQ ID NO:65) based on the referencecrystallographic structures of S. mutans (3AIE, SEQ ID NO:66) (FIG. 8A)and L. reuteri (3KLK, SEQ ID NO:67) (FIG. 8BA). The main chain foldingof the homology model in each view is shown with dark lines while themain chain folding of the reference structure is shown with lighterlines. The residues forming the catalytic sites in the referencecrystallographic structures are shown as Van der Waals spheres forreference. Motif 3a (between the arrows) is presented in both homologymodels as an open loop (black) extending into the solvent as aconsequence of there being no homologous segment to provide means toposition with respect to the remainder of the GTF catalytic domain.

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers Nucleic acidProtein Description SEQ ID NO. SEQ ID NO.″0874 GTF″, Streptococcus sobrinus. DNA codon-  1  2optimized for expression in E. coli. The first 156 (1435 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 450874; a start methionine is included.″6855 GTF″, Streptococcus salivarius SK126. DNA  3  4codon-optimized for expression in E. coli. The first (1341 aa)178 amino acids of the protein are deleted comparedto GENBANK Identification No. 228476855; a start methionine is included.″2379 GTF″, Streptococcus salivarius. DNA codon-  5  6optimized for expression in E. coli. The first 203 (1247 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 662379; a start methionine is included.″7527″ or ″GTFJ″, Streptococcus salivarius. DNA  7  8codon-optimized for expression in E. coli. The first 42 (1477 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 47527; a start methionine is included.″1724 GTF″, Streptococcus downei. DNA codon-  9 10optimized for expression in E. coli. The first 162 (1436 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 121724; a start methionine is included.″0544 GTF″, Streptococcus mutans. DNA codon- 11 12optimized for expression in E. coli. The first 164 (1313 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 290580544; a start methionine is included.″5926 GTF″, Streptococcus dentirousetti. DNA 13 14codon-optimized for expression in E. coli. The first (1323 aa)144 amino acids of the protein are deleted comparedto GENBANK Identification No. 167735926; a start methionine is included.″4297 GTF″, Streptococcus oralis. DNA codon- 15 16optimized for expression in E. coli. The first 228 (1348 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 7684297; a start methionine is included.″5618 GTF″, Streptococcus sanguinis. DNA codon- 17 18optimized for expression in E. coli. The first 223 (1348 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 328945618; a start methionine is included.″2765 GTF″, unknown Streptococcus sp. 0150. DNA 19 20codon-optimized for expression in E. coli. The first (1340 aa)193 amino acids of the protein are deleted comparedto GENBANK Identification No. 322372765; a start methionine is included.″4700 GTF″, Leuconostoc mesenteroides. DNA 21 22codon-optimized for expression in E. coli. The first 36 (1492 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 21654700; a start methionine is included.″1366 GTF″, Streptococcus criceti. DNA codon- 23 24optimized for expression in E. coli. The first 139 (1323 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 146741366; a start methionine is included.″0427 GTF″, Streptococcus sobrinus. DNA codon- 25 26optimized for expression in E. coli. The first 156 (1435 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 940427; a start methionine is included.″2919 GTF″, Streptococcus salivarius PS4. DNA 27 28codon-optimized for expression in E. coli. The first 92 (1340 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 383282919; a start methionine is included.″2678 GTF″, Streptococcus salivarius K12. DNA 29 30codon-optimized for expression in E. coli. The first (1341 aa)188 amino acids of the protein are deleted comparedto GENBANK Identification No. 400182678; a start methionine is included.″2381 GTF″, Streptococcus salivarius. DNA codon- 31 32optimized for expression in E. coli. The first 273 (1305 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 662381; a start methionine is included.″3929 GTF″, Streptococcus salivarius JIM8777. DNA 33 34codon-optimized for expression in E. coli. The first (1341 aa)178 amino acids of the protein are deleted comparedto GENBANK Identification No. 387783929; a start methionine is included.″6907 GTF″, Streptococcus salivarius SK126. DNA 35 36codon-optimized for expression in E. coli. The first (1331 aa)161 amino acids of the protein are deleted comparedto GENBANK Identification No. 228476907; a start methionine is included.″6661 GTF″, Streptococcus salivarius SK126. DNA 37 38codon-optimized for expression in E. coli. The first (1305 aa)265 amino acids of the protein are deleted comparedto GENBANK Identification No. 228476661; a start methionine is included.″0339 GTF″, Streptococcus gallolyticus ATCC 43143. 39 40DNA codon-optimized for expression in E. coli. The (1310 aa)first 213 amino acids of the protein are deletedcompared to GENBANK Identification No. 334280339;a start methionine is included.″0088 GTF″, Streptococcus mutans. DNA codon- 41 42optimized for expression in E. coli. The first 189 (1267 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 3130088; a start methionine is included.″9358 GTF″, Streptococcus mutans UA159. DNA 43 44codon-optimized for expression in E. coli. The first (1287 aa)176 amino acids of the protein are deleted comparedto GENBANK Identification No. 24379358; a start methionine is included.″8242 GTF″, Streptococcus gallolyticus ATCC BAA- 45 462069. DNA codon-optimized for expression in E. coli. (1355 aa)The first 191 amino acids of the protein are deletedcompared to GENBANK Identification No. 325978242;a start methionine is included.″3442 GTF″, Streptococcus sanguinis SK405. DNA 47 48codon-optimized for expression in E. coli. The first (1348 aa)228 amino acids of the protein are deleted comparedto GENBANK Identification No. 324993442; a start methionine is included.″7528 GTF″, Streptococcus salivarius. DNA codon- 49 50optimized for expression in E. coli. The first 173 (1427 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 47528; a start methionine is included.″3279 GTF″, Streptococcus sp. 0150. DNA codon- 51 52optimized for expression in E. coli. The first 178 (1393 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 322373279; a start methionine is included.″6491 GTF″, Leuconostoc citreum KM20. DNA 53 54codon-optimized for expression in E. coli. The first (1262 aa)244 amino acids of the protein are deleted comparedto GENBANK Identification No. 170016491; a start methionine is included.″6889 GTF″, Streptococcus salivarius SK126. DNA 55 56codon-optimized for expression in E. coli. The first (1427 aa)173 amino acids of the protein are deleted comparedto GENBANK Identification No. 228476889; a start methionine is included.″4154 GTF″, Lactobacillus reuteri. DNA codon- 57 58optimized for expression in E. coli. The first 38 amino (1735 aa)acids of the protein are deleted compared toGENBANK Identification No. 51574154.″3298 GTF″, Streptococcus sp. 0150. The first 209 59amino acids of the protein are deleted compared to (1242 aa)GENBANK Identification No. 322373298; a start methionine is included.Wild type GTFJ, Streptococcus salivarius. GENBANK 60Identification No. 47527. (1518 aa)Wild type GTF corresponding to 2678 GTF, 61Streptococcus salivarius K12. (1528 aa)Wild type GTF corresponding to 6855 GTF, 62Streptococcus salivarius SK126. (1518 aa)Wild type GTF corresponding to 2919 GTF, 63Streptococcus salivarius PS4. (1431 aa)Wild type GTF corresponding to 2765 GTF, 64 Streptococcus sp. 0150.(1532 aa) Shorter version of 7527, Streptococcus salivarius, 65(also referred to as ″7527-NT″ herein. The first 178 (1341 aa)amino acids of the protein are deleted compared toGENBANK Identification No. 47527; a start methionine is included.″3AIE″, portion of a GTF from Streptococcus mutans 66as annotated in the Protein Data Bank under pdb (844 aa) entry no. 3AIE.″3KLK″, portion of a GTF from Lactobacillus reuteri as 67annotated in the Protein Data Bank under pdb entry (1039 aa) no. 3KLK.Catalytic active site motif FDxxRxDAxDNV 68 (12 aa)Catalytic active site motif ExWxxxDxxY 69 (10 aa)Catalytic active site motif FxRAHD 70 (6 aa)Catalytic active site motif IxNGYAF 71 (7 aa)Motif SxxRxxN upstream of Motifs 1a and 1b 72 (7 aa)Motif GGxxxLLxNDxDxSNPxVQAExLN downstream 73 of Motifs 1a and 1b (24 aa)Motif WxxxDxxY upstream of Motif 2 74 (8 aa)Motif YxFxRAHD downstream of Motif 2 75 (8 aa)Motif YxxGGQ upstream of Motifs 3a and 3b 76 (6 aa)Motif VRxG downstream of Motifs 3a and 3b 77 (4 aa)Motif 1a: D/N-K-S-I/V-L-D-E-Q-S-D-P-N-H (motif i) 78 (13 aa)Motif 2: N-K-D-G-S-K/T-A-Y-N-E-D-G-T-V/A-K-Q/K- 79S-T-I-G-K-Y-N-E-K-Y-G-D-A-S (motif ii) (30 aa)Motif 3a: L-P-T-D-G-K-M-D-N/K-S-D-V-E-L-Y-R-T- 80 N/S-E (motif iii)(19 aa) Motif 1b: D-S/P-R-F-T-Y/F-N-A/Q/P-N-D-P 81 (11 aa)Motif 3b: I-G-N-G-E 82 (5 aa) Wild type GTF corresponding to 5926 GTF,83 Streptococcus dentirousetti. (1466 aa)″7527-NT-dlS1a″, GTF lacking Motif 1a. DNA codon- 84 85optimized for expression in E. coli. (1325 aa)″7527-NT-dlS2″, GTF lacking Motif 2. DNA codon- 86 87optimized for expression in E. coli. (1311 aa)″7527-NT-dlS3a″, GTF lacking Motif 3a. DNA codon- 88 89optimized for expression in E. coli. (1319 aa)″7527-NT-dlS1a,2″, GTF lacking Motifs 1a and 2. 90 91DNA codon-optimized for expression in E. coli. (1295 aa)″7527-NT-dlS1a,3a″, GTF lacking Motifs 1a and 3a. 92 93DNA codon-optimized for expression in E. coli. (1303 aa)″7527-NT-dlS2,3a″, GTF lacking Motifs 2 and 3a. 94 95DNA codon-optimized for expression in E. coli. (1289 aa)″7527-NT-dlS1a,2,3a″, GTF lacking Motifs 1a, 2 and 96 973a. DNA codon-optimized for expression in E. coli. (1273 aa)

DETAILED DESCRIPTION

The disclosures of all patent and non-patent literature cited herein areincorporated herein by reference in their entirety.

Unless otherwise disclosed, the terms “a” and “an” as used herein areintended to encompass one or more (i.e., at least one) of a referencedfeature.

Where present, all ranges are inclusive and combinable, except asotherwise noted. For example, when a range of “1 to 5” is recited, therecited range should be construed as including ranges “1 to 4”, “1 to3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

The terms “alpha-glucan”, “alpha-glucan polymer” and the like are usedinterchangeably herein. An alpha-glucan is a polymer comprising glucosemonomeric units linked together by alpha-glycosidic linkages.

The terms “branched alpha-glucan”, “branched alpha-glucan polymer” andthe like are used interchangeably herein. A branched alpha-glucan insome aspects can have an intrinsic viscosity and/or branching index thatis reduced by at least about 30% compared to poly alpha-1,3-glucan thatis completely or mostly unbranched. A branched alpha-glucan is believedto contain at least both alpha-1,3 and alpha-1,6 glycosidic linkages(e.g., less than 95% alpha-1,3 glycosidic linkages, and more than 5%alpha-1,6-glycosidic linkages), for example. In some aspects, a branchpoint occurs on average at least every 5 monosaccharide units in abranched alpha-glucan herein.

The terms “glycosidic linkage”, “glycosidic bond” and the like are usedinterchangeably herein and refer to the covalent bond that joins acarbohydrate (sugar) molecule to another group such as anothercarbohydrate. The term “alpha-glycosidic linkage” as used herein refersto the type of glycosidic linkage that joins alpha-D-glucose moleculesto each other. The glycosidic linkages of an alpha-glucan herein canalso be referred to as “glucosidic linkages”. Herein, “alpha-D-glucose”will be referred to as “glucose”.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and thelike are used interchangeably herein. Poly alpha-1,3-glucan hereincomprises at least 95% alpha-1,3-glycosidic linkages. Polyalpha-1,3-glucan that comprises 95%, 96%, 97%, 98%, or 99%alpha-1,3-glycosidic linkages is expected to be mostly unbranched, andthat comprising 100% alpha-1,3-glycosidic linkages is linear/unbranched.

The term “intrinsic viscosity” as used herein refers to a measure of thecontribution of a glucan polymer (e.g., branched alpha-glucan) to theviscosity of a liquid (e.g., solution) comprising the glucan polymer.Intrinsic viscosity can be measured, for example using the methodologydisclosed in the Examples below, or as disclosed by Weaver et al. (J.Appl. Polym. Sci. 35:1631-1637) and Chun and Park (Macromol. Chem. Phys.195:701-711), for example.

The terms “branching index”, “branching ratio” and the like (can bedenoted as g′) are used interchangeably herein, and refer to the ratioof hydrodynamic volume of a branched polymer chain with a given molarmass, to the hydrodynamic volume of a linear polymer chain with the samemolar mass. Branched polymer has a smaller size in solution than itslinear counterpart with the same molar mass. Thus, the branching ratiois a useful measure of the overall branching frequency in apolydispersed polymer. Branching index can be measured, for exampleusing the methodology disclosed in the Examples below, or as disclosedby Zdunek et al. (Food Bioprocess Technol. 7:3525-3535) and Herget etal. (BMC Struct. Biol. 8:35).

The term “sucrose” herein refers to a non-reducing disaccharide composedof an alpha-D-glucose molecule and a beta-D-fructose molecule linked byan alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.

The terms “glucosyltransferase enzyme”, “GTF enzyme”, “GTF”,“glucansucrase” and the like are used interchangeably herein. Theactivity of a GTF enzyme herein catalyzes the reaction of the substratesucrose to make the product alpha-glucan and fructose. Other products(byproducts) of a GTF reaction can include glucose, various solublegluco-oligosaccharides (DP2-DP7), and leucrose. Wild type forms of GTFenzymes generally contain (in the N-terminal to C-terminal direction) asignal peptide, a variable domain, a catalytic domain, and aglucan-binding domain. A GTF herein is classified under the glycosidehydrolase family 70 (GH70) according to the CAZy (Carbohydrate-ActiveEnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238,2009).

The term “glucosyltransferase catalytic domain” herein refers to thedomain of a glucosyltransferase enzyme that providesalpha-glucan-synthesizing activity to a glucosyltransferase enzyme. Aglucosyltransferase catalytic domain preferably does not require thepresence of any other domains to have this activity.

The term “parent glucosyltransferase” herein refers to aglucosyltransferase comprising a catalytic domain having (a) an aminoacid sequence that is at least 90% identical to amino acid positions54-957 of SEQ ID NO:65, positions 55-960 of SEQ ID NO:30, positions55-960 of SEQ ID NO:4, positions 55-960 of SEQ ID NO:28, and/orpositions 55-960 of SEQ ID NO:20, and (b) a motif comprising an aminoacid sequence that is at least 90% identical to SEQ ID NO:78, a motifcomprising an amino acid sequence that is at least 90% identical to SEQID NO:79, and a motif comprising an amino acid sequence that is at least90% identical to SEQ ID N0:80. A parent glucosyltransferase hereintypically synthesizes poly alpha-1,3-glucan.

A “reaction solution” as used herein generally refers to a solutioncomprising sucrose, water, at least one active glucosyltransferaseenzyme, and optionally other components. A reaction solution canalternatively be referred to herein as a “glucan synthesis reaction”,“glucan reaction”, “GTF reaction”, or “reaction composition”, forexample. Other components that can be in a glucan synthesis reactioninclude fructose, glucose, leucrose, and soluble gluco-oligosaccharides(e.g., DP2-DP7). It is in a reaction solution where the step ofcontacting water, sucrose and a glucosyltransferase enzyme is performed.The term “under suitable reaction conditions” as used herein refers toreaction conditions that support conversion of sucrose to polyalpha-glucan via glucosyltransferase enzyme activity. A reactionsolution as claimed herein is not believed to be naturally occurring.

The “percent dry solids” of a reaction solution refers to the wt % ofall the sugars in the glucan synthesis reaction. The percent dry solidsof a reaction solution can be calculated, for example, based on theamount of sucrose used to prepare the reaction.

The “yield” of alpha-glucan by a reaction solution herein represents theweight of alpha-glucan product expressed as a percentage of the weightof sucrose substrate that is converted in the reaction. For example, if100 g of sucrose in a reaction solution is converted to products, and 10g of the products is alpha-glucan, the yield of the alpha-glucan wouldbe 10%. This yield calculation can be considered as a measure ofselectivity of the reaction toward alpha-glucan.

The term “motif” herein refers to a distinctive and recurring structuralunit, such as within an amino acid sequence. By “recurring” it is meantthat a motif occurs in multiple related polypeptides, for example.

The term “motif (i)” as used herein refers to an amino acid sequencecomprising a sequence that is at least 90% identical to SEQ ID NO:78(Motif 1a, Table 1).

The term “motif (ii)” as used herein refers to an amino acid sequencecomprising a sequence that is at least 90% identical to SEQ ID NO:79(Motif 2, Table 1).

The term “motif (iii)” as used herein refers to an amino acid sequencecomprising a sequence that is at least 90% identical to SEQ ID NO:80(Motif 3a, Table 1).

The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” andthe like are used interchangeably herein. The percent by volume of asolute in a solution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)”,“weight-weight percentage (% w/w)” and the like are used interchangeablyherein. Percent by weight refers to the percentage of a material on amass basis as it is comprised in a composition, mixture, or solution.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, “nucleotide sequence” and the like are used interchangeablyherein. A polynucleotide may be a polymer of DNA or RNA that is single-or double-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide may be comprised of one ormore segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.Nucleotides (ribonucleotides or deoxyribonucleotides) can be referred toby a single letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate (for RNA or DNA, respectively), “G” for guanylate ordeoxyguanylate (for RNA or DNA, respectively), “U” for uridylate (forRNA), “T” for deoxythymidylate (for DNA), “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, “W” for A or T, and “N” for any nucleotide (e.g., N can be A,C, T, or G, if referring to a DNA sequence; N can be A, C, U, or G, ifreferring to an RNA sequence).

The term “gene” as used herein refers to a DNA polynucleotide sequencethat expresses an RNA (RNA is transcribed from the DNA polynucleotidesequence) from a coding region, which RNA can be a messenger RNA(encoding a protein) or a non-protein-coding RNA. A gene may refer tothe coding region alone, or may include regulatory sequences upstreamand/or downstream to the coding region (e.g., promoters, 5′-untranslatedregions, 3′-transcription terminator regions). A coding region encodinga protein can alternatively be referred to herein as an “open readingframe” (ORF). A gene that is “native” or “endogenous” refers to a geneas found in nature with its own regulatory sequences; such a gene islocated in its natural location in the genome of a host cell. A“chimeric” gene refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature(i.e., the regulatory and coding regions are heterologous with eachother). Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A“foreign” or “heterologous” gene refers to a gene that is introducedinto the host organism by gene transfer. Foreign/heterologous genes cancomprise native genes inserted into a non-native organism, native genesintroduced into a new location within the native host, or chimericgenes. Polynucleotide sequences in certain embodiments herein areheterologous. A “transgene” is a gene that has been introduced into thegenome by a gene delivery procedure (e.g., transformation). A“codon-optimized” open reading frame has its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

A “non-native” amino acid sequence or polynucleotide sequence hereincomprised in a cell or organism herein does not occur in a native(natural) counterpart of such cell or organism.

“Regulatory sequences” as used herein refer to nucleotide sequenceslocated upstream of a gene's transcription start site (e.g., promoter),5′ untranslated regions, introns, and 3′ non-coding regions, and whichmay influence the transcription, processing or stability, and/ortranslation of an RNA transcribed from the gene. Regulatory sequencesherein may include promoters, enhancers, silencers, 5′ untranslatedleader sequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, stem-loop structures, andother elements involved in regulation of gene expression. One or moreregulatory elements herein may be heterologous to a coding regionherein.

A “promoter” as used herein refers to a DNA sequence capable ofcontrolling the transcription of RNA from a gene. In general, a promotersequence is upstream of the transcription start site of a gene.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. Promoters that cause agene to be expressed in a cell at most times under all circumstances arecommonly referred to as “constitutive promoters”. One or more promotersherein may be heterologous to a coding region herein.

A “strong promoter” as used herein refers to a promoter that can directa relatively large number of productive initiations per unit time,and/or is a promoter driving a higher level of gene transcription thanthe average transcription level of the genes in a cell.

The terms “3′ non-coding sequence”, “transcription terminator”,“terminator” and the like as used herein refer to DNA sequences locateddownstream of a coding sequence. This includes polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression.

As used herein, a first nucleic acid sequence is “hybridizable” to asecond nucleic acid sequence when a single-stranded form of the firstnucleic acid sequence can anneal to the second nucleic acid sequenceunder suitable annealing conditions (e.g., temperature, solution ionicstrength). Hybridization and washing conditions are well known andexemplified in Sambrook J, Fritsch E F and Maniatis T, MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1989), which is incorporated herein byreference, particularly Chapter 11 and Table 11.1

The term “DNA manipulation technique” refers to any technique in whichthe sequence of a DNA polynucleotide sequence is modified. Although theDNA polynucleotide sequence being modified can be used as a substrateitself for modification, it does not have to be physically in hand forcertain techniques (e.g., a sequence stored in a computer can be used asthe basis for the manipulation technique). A DNA manipulation techniquecan be used to delete and/or mutate one or more DNA sequences in alonger sequence. Examples of a DNA manipulation technique includerecombinant DNA techniques (restriction and ligation, molecularcloning), polymerase chain reaction (PCR), and synthetic DNA methods(e.g., oligonucleotide synthesis and ligation). Regarding synthetic DNAtechniques, a DNA manipulation technique can entail observing a DNApolynucleotide in silico, determining desired modifications (e.g., oneor more deletions) of the DNA polynucleotide, and synthesizing a DNApolynucleotide that contains the desired modifications.

The term “in silico” herein means in or on an information storage and/orprocessing device such as a computer; done or produced using computersoftware or simulation, i.e., virtual reality.

The terms “cassette”, “expression cassette”, “gene cassette” and thelike are used interchangeably herein. A cassette can refer to a promoteroperably linked to a DNA sequence encoding a protein-coding RNA. Acassette may optionally be operably linked to a 3′ non-coding sequence.The structure of a cassette herein can optionally be represented by thesimple notation system of “X::Y::Z”. Specifically, X describes apromoter, Y describes a coding sequence, and Z describes a terminator(optional); X is operably linked to Y, and Y is operably linked to Z.

The terms “upstream” and “downstream” as used herein with respect topolynucleotides refer to “5′ of” and “3′ of”, respectively.

The term “expression” as used herein refers to (i) transcription of RNA(e.g., mRNA or a non-protein-coding RNA) from a coding region, and/or(ii) translation of a polypeptide from mRNA. Expression of a codingregion of a polynucleotide sequence can be up-regulated ordown-regulated in certain embodiments.

The term “operably linked” as used herein refers to the association oftwo or more nucleic acid sequences such that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence when it is capable of affecting the expression of thatcoding sequence. That is, the coding sequence is under thetranscriptional control of the promoter. A coding sequence can beoperably linked to one (e.g., promoter) or more (e.g., promoter andterminator) regulatory sequences, for example.

The term “recombinant” when used herein to characterize a DNA sequencesuch as a plasmid, vector, or construct refers to an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis and/or by manipulation of isolated segments ofnucleic acids by genetic engineering techniques. Methods for preparingrecombinant constructs/vectors herein can follow standard recombinantDNA and molecular cloning techniques as described by J. Sambrook and D.Russell (Molecular Cloning: A Laboratory Manual, 3rd Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); T. J.Silhavy et al. (Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1984); and F. M. Ausubel etal. (Short Protocols in Molecular Biology, 5th Ed. Current Protocols,John Wiley and Sons, Inc., NY, 2002), for example.

The term “transformation” as used herein refers to the transfer of anucleic acid molecule into a host organism or host cell by any method. Anucleic acid molecule that has been transformed into an organism/cellmay be one that replicates autonomously in the organism/cell, or thatintegrates into the genome of the organism/cell, or that existstransiently in the cell without replicating or integrating. Non-limitingexamples of nucleic acid molecules suitable for transformation aredisclosed herein, such as plasmids and linear DNA molecules. Hostorganisms/cells herein containing a transforming nucleic acid sequencecan be referred to as “transgenic”, “recombinant”, “transformed”,“engineered”, as a “transformant”, and/or as being “modified forexogenous gene expression”, for example.

The terms “sequence identity” or “identity” as used herein with respectto polynucleotide or polypeptide sequences refer to the nucleic acidbases or amino acid residues in two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.Thus, “percentage of sequence identity” or “percent identity” refers tothe value determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the results by 100to yield the percentage of sequence identity. It would be understoodthat, when calculating sequence identity between a DNA sequence and anRNA sequence, T residues of the DNA sequence align with, and can beconsidered “identical” with, U residues of the RNA sequence. Forpurposes of determining “percent complementarity” of first and secondpolynucleotides, one can obtain this by determining (i) the percentidentity between the first polynucleotide and the complement sequence ofthe second polynucleotide (or vice versa), for example, and/or (ii) thepercentage of bases between the first and second polynucleotides thatwould create canonical Watson and Crick base pairs.

The Basic Local Alignment Search Tool (BLAST) algorithm, which isavailable online at the National Center for Biotechnology Information(NCBI) website, may be used, for example, to measure percent identitybetween or among two or more of the polynucleotide sequences (BLASTNalgorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein.Alternatively, percent identity between sequences may be performed usinga Clustal algorithm (e.g., ClustalW, ClustalV, or Clustal-Omega). Formultiple alignments using a Clustal method of alignment, the defaultvalues may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10.Default parameters for pairwise alignments and calculation of percentidentity of protein sequences using a Clustal method may be KTUPLE=1,GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, theseparameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4. Alternatively still, percent identity between sequences may beperformed using an EMBOSS algorithm (e.g., needle) with parameters suchas GAP OPEN=10, GAP EXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10,END GAP EXTEND=0.5 using a BLOSUM matrix (e.g., BLOSUM62).

Various polypeptide amino acid sequences and polynucleotide sequencesare disclosed herein as features of certain embodiments. Variants ofthese sequences that are at least about 70-85%, 85-90%, or 90%-95%identical to the sequences disclosed herein can be used or referenced.Alternatively, a variant amino acid sequence or polynucleotide sequencecan have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. Thevariant amino acid sequence or polynucleotide sequence has the samefunction/activity of the disclosed sequence, or at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of the function/activity of the disclosedsequence. Any polypeptide amino acid sequence disclosed herein notbeginning with a methionine can typically further comprise at least astart-methionine at the N-terminus of the amino acid sequence.

All the amino acid residues at each amino acid position of the proteinsdisclosed herein are examples. Given that certain amino acids sharesimilar structural and/or charge features with each other (i.e.,conserved), the amino acid at each position of a protein herein can beas provided in the disclosed sequences or substituted with a conservedamino acid residue (“conservative amino acid substitution”) as follows:

-   -   1. The following small aliphatic, nonpolar or slightly polar        residues can substitute for each other: Ala (A), Ser (S), Thr        (T), Pro (P), Gly (G);    -   2. The following polar, negatively charged residues and their        amides can substitute for each other: Asp (D), Asn (N), Glu (E),        Gln (Q);    -   3. The following polar, positively charged residues can        substitute for each other: His (H), Arg (R), Lys (K);    -   4. The following aliphatic, nonpolar residues can substitute for        each other: Ala (A), Leu (L), Ile (I), Val (V), Cys (C), Met        (M); and    -   5. The following large aromatic residues can substitute for each        other: Phe (F), Tyr (Y), Trp (W).

The term “isolated” as used herein refers to a polynucleotide orpolypeptide molecule that has been completely or partially purified fromits native source. In some instances, the isolated polynucleotide orpolypeptide molecule is part of a greater composition, buffer system orreagent mix. For example, the isolated polynucleotide or polypeptidemolecule can be comprised within a cell or organism in a heterologousmanner. “Isolated” herein can also characterize embodiments that aresynthetic/man-made, and/or have properties that are not naturallyoccurring.

The term “increased” as used herein can refer to a quantity or activitythat is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% morethan the quantity or activity for which the increased quantity oractivity is being compared. The terms “increased”, “elevated”,“enhanced”, “greater than”, “improved” and the like are usedinterchangeably herein.

Some advances have been made in producing linear glucan polymers havinga high percentage of alpha-1,3 glycosidic linkages suitable for use inspinning fibers. However, less attention appears to have been drawn toproducing branched alpha-glucan polymers.

Thus, disclosed herein are glucosyltransferases that can synthesizebranched alpha-glucan. Some embodiments disclosed herein concern aglucosyltransferase enzyme comprising a catalytic domain that comprisesan amino acid sequence that is at least 90% identical to amino acidpositions 54-941 of SEQ ID NO:85, 54-927 of SEQ ID NO:87, 54-935 of SEQID NO:89, 54-911 of SEQ ID NO:91, 54-919 of SEQ ID NO:93, 54-905 of SEQID NO:95, or 54-889 of SEQ ID NO:97, wherein the catalytic domain lacksat least one motif selected from the group consisting of:

-   (i) a motif comprising an amino acid sequence that is at least 90%    identical to SEQ ID NO:78,-   (ii) a motif comprising an amino acid sequence that is at least 90%    identical to SEQ ID NO:79, and-   (iii) a motif comprising an amino acid sequence that is at least 90%    identical to SEQ ID NO:80;    and wherein the glucosyltransferase enzyme produces a branched    alpha-glucan polymer.

A glucosyltransferase enzyme herein, since it lacks one or more ofmotifs (i), (ii), and/or (iii), can optionally be characterized as amodified glucosyltransferase enzyme. Such a modified glucosyltransferaseproduces branched alpha-glucan polymer by virtue of lacking one or moreof the above motifs. In contrast, a glucosyltransferase that has acatalytic domain comprising an amino acid sequence of at least 90%identity to amino acid positions 54-957 of SEQ ID NO:65 and that has allthree of the above motifs can produce poly alpha-1,3-glucan having atleast 95% alpha-1,3-linkages (such a glucan polymer is mostly orcompletely linear). Note that each of the above portions of SEQ IDNOs:85, 87, 89, 91, 93, 95 and 97 can be derived from amino acidpositions 54-957 of SEQ ID NO:65 (refer to Examples 6-11), but in amanner lacking motifs i, ii, and/or iii. For example, consider that:

residues 54-941 of SEQ ID NO:85 essentially represent positions 54-957of SEQ ID NO:65, but in which motif (i) is lacking;

residues 54-927 of SEQ ID NO:87 essentially represent positions 54-957of SEQ ID NO:65, but in which motif (ii) is lacking;

residues 54-935 of SEQ ID NO:89 essentially represent positions 54-957of SEQ ID NO:65, but in which motif (iii) is lacking;

residues 54-911 of SEQ ID NO:91 essentially represent positions 54-957of SEQ ID NO:65, but in which motifs (i) and (ii) are lacking;

residues 54-919 of SEQ ID NO:93 essentially represent positions 54-957of SEQ ID NO:65, but in which motifs (i) and (iii) are lacking;

residues 54-905 of SEQ ID NO:95 essentially represent positions 54-957of SEQ ID NO:65, but in which motifs (ii) and (iii) are lacking; and

residues 54-889 of SEQ ID NO:97 essentially represent positions 54-957of SEQ ID NO:65, but in which motif (i), (ii) and (iii) are lacking;

The catalytic domain of a glucosyltransferase as presently disclosed cancomprise an amino acid sequence that is at least 90% identical to aminoacid positions 54-941 of SEQ ID NO:85, 54-927 of SEQ ID NO:87, 54-935 ofSEQ ID NO:89, 54-911 of SEQ ID NO:91, 54-919 of SEQ ID NO:93, 54-905 ofSEQ ID NO:95, or 54-889 of SEQ ID NO:97. In certain embodiments, theamino acid sequence of a glucosyltransferase catalytic domain can be atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%identical to amino acid positions 54-941 of SEQ ID NO:85, 54-927 of SEQID NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQ ID NO:91, 54-919 of SEQID NO:93, 54-905 of SEQ ID NO:95, or 54-889 of SEQ ID NO:97.

Amino acid positions 54-957 of SEQ ID NO:65 represent, approximately, acatalytic domain sequence of the glucosyltransferase identified inGENBANK under GI number 47527 (SEQ ID NO:60). SEQ ID NO:65 generallyrepresents the catalytic domain and glucan-binding domain of SEQ IDNO:60; the signal peptide and variable domains are missing from SEQ IDNO:65. As shown in Example 14, a catalytic domain sequence of SEQ IDNO:65 (residues 54-957) was able to catalyze the production of analpha-glucan. Example 14 also shows that a catalytic domain sequence ofSEQ ID NO:14 (residues 57-906 of SEQ ID NO:14 [GTF 5926]) was able tocatalyze production of an alpha-glucan. The molecular weight of thealpha-glucan produced by each of these catalytic domain sequencesgenerally corresponded with the molecular weight of the product producedby their enzyme counterparts containing both the catalytic domain andglucan binding domain (refer to activity of SEQ ID NOs:65 and 14 inTable 4, DP_(w)150). Thus, it is believed that a catalytic domainsequence herein is an important structural component for aglucosyltransferase enzyme to be capable of producing alpha-glucanpolymer.

Although it is believed that a glucosyltransferase enzyme herein needonly have a catalytic domain sequence comprising an amino acid sequencethat is at least 90% identical to amino acid positions 54-941 of SEQ IDNO:85, 54-927 of SEQ ID NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQ IDNO:91, 54-919 of SEQ ID NO:93, 54-905 of SEQ ID NO:95, or 54-889 of SEQID NO:97 (and lacking motif i, ii, and/or iii), the glucosyltransferaseenzyme can be comprised within a larger amino acid sequence. Forexample, the catalytic domain may be linked at its C-terminus to aglucan-binding domain, and/or linked at its N-terminus to a variabledomain and/or signal peptide. Examples of glucosyltransferase enzymesherein comprising catalytic and glucan-binding domains can comprise SEQID NO:85, 87, 89, 91, 93, 95, or 97, or an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or99.5% identical to any of these sequences (and lacking motif i, ii,and/or iii).

Still further examples of glucosyltransferase enzymes can be any asdisclosed herein and that include 1-300 (or any integer there between[e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on theN-terminus and/or C-terminus. Such additional residues may be from acorresponding wild type sequence from which the glucosyltransferaseenzyme is derived, or may be a heterologous sequence such as an epitopetag (at either N- or C-terminus) or a heterologous signal peptide (atN-terminus), for example. Examples include glucosyltransferase enzymescomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to SEQ ID NO:65,30, 4, 28, or 20, and that lack motif i, ii, and/or iii. These sequences(SEQ ID NO:65, 30, 4, 28, 20) lack an N-terminal signal peptide (as wellas a variable domain) (refer to Table 1). Still other examples includeglucosyltransferase enzymes that (i) comprise an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%,99%, or 99.5% A identical to SEQ ID NO:60, 61, 62, 63, or 64, and (i)lack motif i, ii, and/or iii.

An N-terminal start-methionine (amino acid position 1) has been added tocertain sequences herein for intracellular expression purposes(expressed enzyme can be obtained in a cell lysate, for example) (e.g.,SEQ ID NOs:85, 87, 89, 91, 93, 95, 97, 65, 30, 4, 28, 20). One of skillin the art would understand that an intervening heterologous amino acidsequence such as an epitope and/or signal peptide could optionally beadded between the start methionine and glucosyltransferase sequence.Thus, for example, a glucosyltransferase enzyme herein may comprise anamino acid sequence that (i) is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to the amino acid sequencebeginning at position 2 of a disclosed amino acid sequence, and (ii)lacks motif i, ii, and/or iii.

A glucosyltransferase enzyme herein typically lacks an N-terminal signalpeptide. An expression system for producing a glucosyltransferase enzymeherein may employ an enzyme-encoding polynucleotide that furthercomprises sequence encoding an N-terminal signal peptide to directextra-cellular secretion, if desired. The signal peptide in suchembodiments is cleaved from the enzyme during the secretion process. Thesignal peptide may either be native or heterologous to theglucosyltransferase. An example of a signal peptide useful herein is onefrom a bacterial (e.g., a Bacillus species such as B. subtilis) orfungal species. An example of a bacterial signal peptide is an aprEsignal peptide, such as one from Bacillus (e.g., B. subtilis, seeVogtentanz et al., Protein Expr. Purif. 55:40-52, which is incorporatedherein by reference).

FIGS. 2A-O show that a catalytic domain sequence of GTF 7527 (residues54-957 of SEQ ID NO:65) aligns with catalytic domain sequences ofseveral other glucosyltransferase enzymes, with several regions showingcomplete conservation across all the sequences (residues with darkbackground). The dark background residues in FIGS. 2A-O visually map outthe catalytic domain of each sequence, indicating their length to beabout 850 to 900 amino acid residues long. Thus, the catalytic domain ofa glucosyltransferase enzyme herein can be about 790 to 840, 850 to 900,or 790 to 900 (or any integer between 790 and 900) amino acid residueslong (some of these numbers take into account embodiments in whichmotifs i, iii, and/or iii are removed), for example.

Certain of the conserved regions in FIGS. 2A-O include catalytic activesite motifs SEQ ID NOs:68, 69, 70, and 71 (refer to Example 3). Thus, acatalytic domain sequence of a glucosyltransferase enzyme in someaspects can contain one or more of SEQ ID NOs:68, 69, 70, and 71 inalignment, respectively, with SEQ ID NOs:68, 69, 70, and 71 as presentin amino acids 54-957 of SEQ ID NO:65. Other conserved regions in FIGS.2A-O include SEQ ID NOs:72, 73, 74, 75, 76 and 77 (refer to Example 4).Thus, a catalytic domain sequence of a glucosyltransferase enzyme insome aspects can contain one or more of SEQ ID NOs:72, 73, 74, 75, 76and 77 in alignment, respectively, with SEQ ID NOs:72, 73, 74, 75, 76and 77 as present in amino acids 54-957 of SEQ ID NO:65.

The catalytic domain of a glucosyltransferase enzyme herein can haveactivity as exhibited by a catalytic domain of a glucosyltransferaseclassified under the glycoside hydrolase family 70 (GH70). Such a GH70glucosyltransferase may be found in the CAZy (Carbohydrate-ActiveEnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238,2009), for example.

A glucosyltransferase enzyme herein lacks at least one of motifs (i),(ii), or (iii). Motif (i) corresponds with “Motif 1a” (FIG. 3). Motif(ii) corresponds with “Motif 2” (FIG. 5). Motif (iii) corresponds with“Motif 3a” (FIG. 7). A glucosyltransferase can “lack” one or more ofthese motifs by virtue of a deletion and/or mutation (e.g., amino acidsubstitution), for example. In some embodiments, a glucosyltransferasecan be characterized as lacking one of these motifs if no amino acidsequence within a catalytic domain sequence can be identified to have90% or more identity to SEQ ID NO:78 (motif i), SEQ ID NO:79 (motif ii),or SEQ ID NO:78 (motif iii).

In certain embodiments, motif (i) can be at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:78. Incertain embodiments, motif (ii) can be at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:79. In certainembodiments, motif (iii) can be at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:80. Thus, it can beseen that in some aspects, motif (i) can comprise SEQ ID NO:78, motif(ii) can comprise SEQ ID NO:79, and motif (iii) can comprise SEQ IDNO:80.

Regarding motif (i) in certain embodiments, the first residue of SEQ IDNO:78 (D/N-K-S-IN-L-D-E-Q-S-D-P-N-H) can be an aspartate (D) and thefourth residue can be an isoleucine (I). Alternatively, the firstresidue can be an aspartate (D) and the fourth residue can be a valine(V), or the first residue can be an asparagine (N) and the fourthresidue can be an isoleucine (I), or the first residue can be anasparagine (N) and the fourth residue can be a valine (V).

Regarding motif (ii) in certain embodiments, the sixth residue of SEQ IDNO:79(N-K-D-G-S-K/T-A-Y-N-E-D-G-T-V/A-K-Q/K-S-T-I-G-K-Y-N-E-K-Y-G-D-A-S) canbe a lysine (K), the fourteenth residue can be a valine (V), and thesixteenth residue can be a glutamine (Q). Alternatively, the sixthresidue can be a lysine (K), the fourteenth residue can be an alanine(A), and the sixteenth residue can be a glutamine (Q); or the sixthresidue can be a lysine (K), the fourteenth residue can be an valine(V), and the sixteenth residue can be a lysine (K). Additional examplesinclude where the sixth residue can be a threonine (T).

Regarding motif (iii) in certain embodiments, the ninth residue of SEQID NO:80 (L-P-T-D-G-K-M-D-N/K-S-D-V-E-L-Y-R-T-N/S-E) can be anasparagine (N) and the eighteenth residue can be an asparagine (N).Alternatively, the ninth residue can be an asparagine (N) and theeighteenth residue can be a serine (S), or the ninth residue can be alysine (K) and the eighteenth residue can be an asparagine (N), or theninth residue can be a lysine (K) and the eighteenth residue can be aserine (S).

A glucosyltransferase enzyme as presently disclosed may lack motif (i)only; motif (ii) only; motif (iii) only; both motifs (i) and (ii); bothmotifs (i) and (iii); both motifs (ii) and (iii); and all three ofmotifs (i), (ii) and (iii), for example.

The relative positions of motif (i) (SEQ ID NO:78), motif (ii) (SEQ IDNO:79) and motif (iii) (SEQ ID NO:80) align with residues 231-243,396-425 and 549-567, respectively, of the GTF 7527 sequence (SEQ IDNO:65) shown in FIGS. 2A-O. In certain embodiments herein,

-   -   (A) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:78 in the glucosyltransferase catalytic        domain aligns with amino acid positions 231-243 of SEQ ID NO:65;    -   (B) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:79 in the glucosyltransferase catalytic        domain aligns with amino acid positions 396-425 of SEQ ID NO:65;        and/or    -   (C) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:80 in the glucosyltransferase catalytic        domain aligns with amino acid positions 549-567 of SEQ ID NO:65.        The term “aligns with” can be used interchangeably with        “corresponds to”, “corresponds with”, and the like. The relative        positions of motifs (i), (ii) and/or (iii) in a        glucosyltransferase catalytic domain can thus be determined with        reference to the above amino acid positions in SEQ ID NO:65. For        example, the sequence of a glucosyltransferase catalytic domain        can be aligned with SEQ ID NO:65 using any means known in the        art, such as through use of an alignment algorithm or software        as described above (e.g., BLASTP, ClustalW, ClustalV, EMBOSS).

The relative positions of motifs (i), (ii) and/or (iii) in aglucosyltransferase catalytic domain can be determined with reference tocertain conserved sequences, namely SEQ ID NOs:72, 73, 74, 75, 76 and77, if desired.

Motif 1a (SEQ ID NO:78) is flanked by upstream and downstream conservedsequences as shown in FIG. 3. Preceding Motif 1a is the sequence SxxRxxN(SEQ ID NO:72), and following this motif is the sequenceGGxxxLLxNDxDxSNPxVQAExLN (SEQ ID NO:73). Thus, the position of motif (i)can be located between SEQ ID NOs:72 and 73. SEQ ID NO:72 can bedirectly adjacent (upstream) to motif (i), or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 (or 1-15) amino acid residues upstream motif(i). SEQ ID NO:73 can be directly adjacent (downstream) to motif (i), or1, 2, 3, 4, or 5 (or 1-5) amino acid residues downstream motif (i).

Motif 2 (SEQ ID NO:79) is flanked by upstream and downstream conservedsequences as shown in FIG. 5. Specifically, preceding Motif 2 is thesequence WxxxDxxY (SEQ ID NO:74), and following this motif is thesequence YxFxRAHD (SEQ ID NO:75). Thus, the position of motif (ii) canbe located between SEQ ID NOs:74 and 75. SEQ ID NO:74 can be directlyadjacent (upstream) to motif (ii), or 1-65 (or any integer between 1 and65) amino acid residues upstream motif (ii). SEQ ID NO:75 can bedirectly adjacent (downstream) to motif (ii), or 1, 2, 3, 4, or 5 (or1-5) amino acid residues downstream motif (ii).

Motif 3a (SEQ ID NO:80) is flanked by upstream and downstream conservedsequences as shown in FIG. 7. Specifically, preceding Motif 3a is thesequence YxxGGQ (SEQ ID NO:76), and following this motif is the sequenceVRxG (SEQ ID NO:77). Thus, the position of motif (iii) can be locatedbetween SEQ ID NOs:76 and 77. SEQ ID NO:76 can be directly adjacent(upstream) to motif (iii), or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (or1-11) amino acid residues upstream motif (iii). SEQ ID NO:77 can bedirectly adjacent (downstream) to motif (iii), or 1, 2, 3, 4, 5, 6, 7,8, or 9 (or 1-9) amino acid residues downstream motif (iii).

Certain amino acid positions in the upstream/downstream conservedsequences SEQ ID NOs:72-77 can be any amino acid (indicated by an “x” ineach sequence in Table 1). Examples of SEQ ID NOs:72 and 73 are as shownin any of the GTF sequences in FIGS. 2 and 3 at the amino acids of eachGTF sequence aligning with positions 214-220 and 245-268, respectively,of SEQ ID NO:65 (GTF 7527). Examples of SEQ ID NOs:74 and 75 are asshown in any of the GTF sequences in FIGS. 2 and 5 at the amino acids ofeach GTF sequence aligning with positions 334-341 and 428-435,respectively, of SEQ ID NO:65 (GTF 7527). Examples of SEQ ID NOs:76 and77 are as shown in any of the GTF sequences in FIGS. 2 and 7 at theamino acids of each GTF sequence aligning with positions 537-542 and572-575, respectively, of SEQ ID NO:65 (GTF 7527).

The foregoing location information (e.g., alignment coordinates and/orlocation between certain conserved sequences) can be used, for instance,in an effort to determine whether a glucosyltransferase lacks at leastone of motifs (i), (ii), or (iii).

A glucosyltransferase enzyme herein lacking at least one of motifs (i),(ii), or (iii) can produce a branched alpha-glucan polymer. In someembodiments, alpha-glucan polymer branching can be gauged usingmeasurements of intrinsic viscosity and/or branching index (g′), whichcan be measured following any means known in the art. For example, it isbelieved that Weaver et al. (J. Appl. Polym. Sci. 35:1631-1637) and Chunand Park (Macromol. Chem. Phys. 195:701-711) describe suitabletechniques of measuring intrinsic viscosity, and Zdunek et al. (FoodBioprocess Technol. 7:3525-3535) and Herget et al. (BMC Struct. Biol.8:35) describe suitable techniques for measuring branching index. Allthese references are incorporated herein by reference. Also, themethodology provided in the below Examples can be used, for example.

Alpha-glucan polymer branching herein can, in some aspects, be judgedwith respect to measurements made against poly alpha-1,3-glucancontaining at least 95%, 96%, 97%, 98%, or 99% alpha-1,3 glycosidiclinkages (such polymer is expected to be mostly unbranched), or 100%alpha-1,3 glycosidic linkages (such polymer is linear/unbranched).Measurements can be with respect to intrinsic viscosity and/or branchingindex, for example. In certain embodiments, alpha-glucan produced by aglucosyltransferase herein can have an intrinsic viscosity and/orbranching index (each measurement per methodology disclosed in belowExamples, for example) that is reduced by at least about 30%, 40%, 50%,60%, 70%, 80%, or 90% compared to poly alpha-1,3-glucan that iscompletely or mostly unbranched.

A branched alpha-glucan polymer herein is believed to contain at leastboth alpha-1,3 and alpha-1,6 glycosidic linkages, for example. Abranched alpha-glucan polymer may possibly further comprise alpha-1,2and/or alpha-1,4 glycosidic linkages in some aspects. There are likelyno beta-glycosidic linkages present. In certain embodiments, branchedalpha-glucan polymer can have less than 95%, 94%, 93%, 92%, 91%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% alpha-1,3glycosidic linkages. A branched alpha-glucan polymer can have at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%alpha-1,6-glycosidic linkages in some aspects. It is contemplated that,in some aspects, a branch point occurs on average every (or at leastevery) 5, 10, 15, 20, 25, 30, 35, or 40 monosaccharide units in abranched alpha-glucan herein.

The glycosidic linkage profile of a glucan polymer herein can bedetermined using any method known in the art. For example, the linkageprofile can be determined using methods that use nuclear magneticresonance (NMR) spectroscopy (e.g., ¹³C NMR or ¹H NMR). These and othermethods that can be used are disclosed in Food Carbohydrates: Chemistry,Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W.Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC,Boca Raton, Fla., 2005), which is incorporated herein by reference.

A branched alpha-glucan polymer in most embodiments is insoluble. Suchinsolubility is observed in aqueous conditions (e.g., solvent comprisingat least 90% water) of generally neutral pH (e.g., between 6-8), forexample.

A glucosyltransferase enzyme herein can be derived from any microbialsource, such as a bacteria or fungus. Examples of bacterialglucosyltransferase enzymes are those derived from a Streptococcusspecies, Leuconostoc species or Lactobacillus species. Examples ofStreptococcus species include S. salivarius, S. sobrinus, S.dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S.sanguinis. Examples of Leuconostoc species include L. mesenteroides, L.amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L.dextranicum and L. fructosum. Examples of Lactobacillus species includeL. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei,L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L.fermentum and L. reuteri.

A glucosyltransferase enzyme herein can be produced by any means knownin the art. For example, a glucosyltransferase enzyme may be producedrecombinantly in a heterologous expression system, such as a microbialheterologous expression system. Examples of heterologous expressionsystems include bacterial (e.g., E. coli such as TOP10 or MG1655;Bacillus sp.) and eukaryotic (e.g., yeasts such as Pichia sp. andSaccharomyces sp.) expression systems.

In certain embodiments, a heterologous gene expression system may be onethat is designed for protein secretion. A glucosyltransferase enzymetypically comprises a signal peptide (signal sequence) in suchembodiments. The signal peptide may be either its native signal peptideor a heterologous signal peptide.

A glucosyltransferase enzyme described herein may be used in anypurification state (e.g., pure or non-pure). For example, aglucosyltransferase enzyme may be purified and/or isolated prior to itsuse. Examples of glucosyltransferase enzymes that are non-pure includethose in the form of a cell lysate. A cell lysate or extract may beprepared from a bacteria (e.g., E. coli) used to heterologously expressthe enzyme. For example, the bacteria may be subjected to disruptionusing a French pressure cell. In alternative embodiments, bacteria maybe homogenized with a homogenizer (e.g., APV, Rannie, Gaulin). Aglucosyltransferase enzyme is typically soluble in these types ofpreparations. A bacterial cell lysate, extract, or homogenate herein maybe used at about 0.15-0.3% (v/v), for example, in a reaction solutionfor producing branched alpha-glucan.

The activity of a glucosyltransferase enzyme herein can be determinedusing any method known in the art. For example, glucosyltransferaseenzyme activity can be determined by measuring the production ofreducing sugars (fructose and glucose) in a reaction solution containingsucrose (50 g/L), dextran T10 (1 mg/mL) and potassium phosphate buffer(pH 6.5, 50 mM), where the solution is held at 22-25° C. for 24-30hours. The reducing sugars can be measured, for instance, by adding 0.01mL of the reaction solution to a mixture containing 1 N NaOH and 0.1%triphenyltetrazolium chloride and then monitoring the increase inabsorbance at OD_(480nm) for five minutes.

Some embodiments disclosed herein concern a polynucleotide comprising anucleotide sequence that encodes a glucosyltransferase as presentlydisclosed (e.g., a GTF comprising a catalytic domain with an amino acidsequence that [i] is at least 90% identical to positions 54-941 of SEQID NO:85, 54-927 of SEQ ID NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQID NO:91, 54-919 of SEQ ID NO:93, 54-905 of SEQ ID NO:95, or 54-889 ofSEQ ID NO:97, and [ii] lacks at least one of motifs i, ii, or iii).Optionally, one or more regulatory sequences are operably linked to thenucleotide sequence, and preferably a promoter sequence is included as aregulatory sequence.

A polynucleotide comprising a nucleotide sequence encoding aglucosyltransferase herein can be a vector or construct useful fortransferring a nucleotide sequence into a cell, for example. Examples ofa suitable vector/construct can be selected from a plasmid, yeastartificial chromosome (YAC), cosmid, phagemid, bacterial artificialchromosome (BAC), virus, or linear DNA (e.g., linear PCR product). Apolynucleotide sequence in some aspects can be capable of existingtransiently (i.e., not integrated into the genome) or stably (i.e.,integrated into the genome) in a cell. A polynucleotide sequence in someaspects can comprise, or lack, one or more suitable marker sequences(e.g., selection or phenotype marker).

A polynucleotide sequence in certain embodiments can comprise one ormore regulatory sequences operably linked to the nucleotide sequenceencoding a glucosyltransferase. For example, a nucleotide sequenceencoding a glucosyltransferase may be in operable linkage with apromoter sequence (e.g., a heterologous promoter). A promoter sequencecan be suitable for expression in a cell (e.g., bacterial cell such asE. coli; eukaryotic cell such as a fungus, yeast, insect, or mammaliancell) or in an in vitro protein expression system, for example. Examplesof other suitable regulatory sequences are disclosed herein (e.g.,transcription terminator sequences).

In some embodiments, a polynucleotide sequence does not comprise aregulatory sequence operably linked to a nucleotide encoding aglucosyltransferase. Such a polynucleotide could be a cloning vector(e.g., cloning plasmid), for example, used simply for sub-cloning orgene shuttling purposes.

A promoter sequence herein can be constitutive or inducible, forexample. A promoter in certain aspects can comprise a strong promoter,which is a promoter that can direct a relatively large number ofproductive initiations per unit time, and/or is a promoter driving ahigher transcription level than the average transcription level of thegenes in a cell comprising the strong promoter. Examples of strongpromoters useful herein include some bacterial and phage promoters thatare well known in the art, and some yeast promoters (e.g., Velculescu etal., Cell 88:243-251, incorporated herein by reference).

The present disclosure also concerns a method of preparing apolynucleotide sequence encoding a glucosyltransferase enzyme thatproduces a branched alpha-glucan polymer. This method comprises:

(a) identifying a polynucleotide sequence encoding a parentglucosyltransferase enzyme that comprises a catalytic domain comprising:

-   -   (1) an amino acid sequence that is at least 90% identical to        amino acid positions 54-957 of SEQ ID NO:65, and    -   (2) the following three motifs:        -   (i) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:78,        -   (ii) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:79, and        -   (iii) a motif comprising an amino acid sequence that is at            least 90% identical to SEQ ID NO:80;            and            (b) modifying the polynucleotide sequence identified in            step (a) to delete and/or mutate at least one of motifs (i),            (ii), or (iii) encoded by the polynucleotide sequence,            thereby providing a polynucleotide sequence encoding a            glucosyltransferase enzyme that produces a branched            alpha-glucan polymer. Examples of a polynucleotide sequence            produced by such a method are disclosed in the above            embodiments concerning a polynucleotide sequence. The            glucosyltransferase encoded by the polynucleotide sequence            produced in step (b) can be characterized as a child            glucosyltransferase, if desired.

Identification step (a) herein can, in some instances, compriseidentifying an amino acid sequence of a parent glucosyltransferaseenzyme. A polynucleotide sequence could be determined from this aminoacid sequence according to the genetic code (codons), such as thegenetic code used in the species from which the parentglucosyltransferase was identified.

The presence of motifs (i), (ii), and (iii) in the catalytic domain of aparent glucosyltransferase enzyme can be detected following any meansknown in the art and/or any procedure described herein. For example,detection can be performed (a) in silico, (b) with a method comprising anucleic acid hybridization step, (c) with a method comprising a proteinsequencing step, and/or (d) with a method comprising a protein bindingstep.

Motifs (i), (ii) and (iii) were identified by in silico detection (seeExample 4 below). Thus, the amino acid sequences of parentglucosyltransferase enzymes (and/or nucleotide sequences encoding suchglucosyltransferase enzymes) stored in a computer or database (e.g.,public databases such as GENBANK, EMBL, REFSEQ, GENEPEPT, SWISS-PROT,PIR, PDB) can be reviewed in silico to identify a glucosyltransferaseenzyme comprising motifs (i), (ii) and (iii) in its catalytic domain,for example. Such review could comprise using any means known in the artsuch as through use of an alignment algorithm or software as describedabove (e.g., BLASTN, BLASTP, ClustalW, ClustalV, EMBOSS). The sequenceof the glucosyltransferase catalytic domain being reviewed could bealigned with a catalytic domain sequence of SEQ ID NO:65 (GTF 7527),which comprises Motifs 1a (SEQ ID NO:78), 2 (SEQ ID NO:79) and 3a (SEQID NO:80), to detect the presence or absence of motifs (i), (ii), and/or(iii). Alternatively, the sequence of the glucosyltransferase catalyticdomain being reviewed could be aligned with a catalytic domain sequenceof SEQ ID NO:30 (GTF 2678), SEQ ID NO:4 (GTF 6855), SEQ ID NO:28 (GTF2919), and/or SEQ ID NO:20 (GTF 2765), all of which comprise Motifs 1a(SEQ ID NO:78), 2 (SEQ ID NO:79) and 3a (SEQ ID NO:80), to identify thepresence or absence of motifs (i), (ii), and/or (iii).

Another in silico means for detecting motifs (i), (ii), and (iii) in aglucosyltransferase catalytic domain sequence can comprise comparing thepredicted three-dimensional structure (tertiary structure) of aglucosyltransferase catalytic domain sequence with a referencestructure. The structures of both the catalytic domain being reviewedand the reference can be visually compared using any means known in theart such as with a computer program that provides a structure based onamino acid sequence input (e.g., software package MOE, ChemicalComputing Group, Montreal, Canada). For example, if the referencestructure lacks motif (i), (ii), and/or (iii), the comparison may detectthe presence of motif (i), (ii), and/or (iii) by showing a domain(s) inthe structure being reviewed that does not have a corresponding domainin the reference structure. Examples of this type of comparison areshown in FIGS. 4a, 4b, 6a, 6b, 8a and 8 b.

Alternatively, identifying a parent glucosyltransferase enzyme havingmotifs (i), (ii), and (iii) in its catalytic domain can be performed viaa method comprising a nucleic acid hybridization step. Such a method cancomprise using DNA hybridization (e.g., Southern blot, dot blot), RNAhybridization (e.g., northern blot), or any other method that has anucleic acid hybridization step (e.g., DNA sequencing, PCR, RT-PCR, allof which may comprise hybridization of an oligonucleotide), for example.As an example, an oligonucleotide that would hybridize to a nucleotidesequence encoding Motif 1a (SEQ ID NO:78), 2 (SEQ ID NO:79), or 3a (SEQID NO:80) could be used to detect its presence or absence in apolynucleotide sequence encoding the glucosyltransferase catalyticdomain being reviewed. Conditions and parameters for carrying outhybridization methods in general are well known and disclosed, forexample, in Sambrook J, Fritsch E F and Maniatis T, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y. (1989); Silhavy T J, Bennan M L and Enquist L W, Experiments withGene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.(1984); Ausubel F M et al., Current Protocols in Molecular Biology,published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken,N.J. (1987); and Innis M A, Gelfand D H, Sninsky J J and White T J(Editors), PCR Protocols: A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif. (1990).

In another aspect, a parent glucosyltransferase enzyme comprising motifs(i), (ii), and (iii) in its catalytic domain can be detected using amethod comprising a protein sequencing step. Such a protein sequencingstep can comprise one or more procedures such as N-terminal amino acidanalysis, C-terminal amino acid analysis, Edman degradation, or massspectrometry, for example.

In still another aspect, a parent glucosyltransferase enzyme comprisingmotifs (i), (ii), and (iii) in its catalytic domain can be detectedusing a method comprising a protein binding step. Such a protein bindingstep could be performed using an antibody that specifically binds to oneof these motifs, for example. Antibodies for identifying the presence orabsence of motif (i) can be specific for an amino acid sequence that isat least 90% identical to SEQ ID NO:78. Antibodies for identifying thepresence or absence of motif (ii) can be specific for an amino acidsequence that is at least 90% identical to SEQ ID NO:79. Antibodies foridentifying the presence or absence of motif (iii) can be specific foran amino acid sequence that is at least 90% identical to SEQ ID NO:80.

A parent glucosyltransferase in a polynucleotide preparation methodherein comprises a catalytic domain comprising motifs (i), (ii) and(iii). Motif (i) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO:78. Motif (ii) can be at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO:79. Motif (iii) can be at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:80. Thus, it can beseen that in certain embodiments of an identification method herein,motif (i) can comprise SEQ ID NO:78, motif (ii) can comprise SEQ IDNO:79, and motif (iii) can comprise SEQ ID NO:80.

Regarding motif (i) in certain embodiments, the first residue of SEQ IDNO:78 (D/N-K-S-IN-L-D-E-Q-S-D-P-N-H) can be an aspartate (D) and thefourth residue can be an isoleucine (I). Alternatively, the firstresidue can be an aspartate (D) and the fourth residue can be a valine(V), or the first residue can be an asparagine (N) and the fourthresidue can be an isoleucine (I), or the first residue can be anasparagine (N) and the fourth residue can be a valine (V).

Regarding motif (ii) in certain embodiments, the sixth residue of SEQ IDNO:79(N-K-D-G-S-K/T-A-Y-N-E-D-G-T-V/A-K-Q/K-S-T-I-G-K-Y-N-E-K-Y-G-D-A-S) canbe a lysine (K), the fourteenth residue can be a valine (V), and thesixteenth residue can be a glutamine (Q). Alternatively, the sixthresidue can be a lysine (K), the fourteenth residue can be an alanine(A), and the sixteenth residue can be a glutamine (Q); or the sixthresidue can be a lysine (K), the fourteenth residue can be an valine(V), and the sixteenth residue can be a lysine (K). Additional examplesinclude where the sixth residue can be a threonine (T).

Regarding motif (iii) in certain embodiments, the ninth residue of SEQID NO:80 (L-P-T-D-G-K-M-D-N/K-S-D-V-E-L-Y-R-T-N/S-E) can be anasparagine (N) and the eighteenth residue can be an asparagine (N).Alternatively, the ninth residue can be an asparagine (N) and theeighteenth residue can be a serine (S), or the ninth residue can be alysine (K) and the eighteenth residue can be an asparagine (N), or theninth residue can be a lysine (K) and the eighteenth residue can be aserine (S).

Any of the above features regarding the location of motifs (i), (ii) and(iii) in a glucosyltransferase enzyme catalytic domain sequence can beused appropriately to detect one or more of these motifs in a parentglucosyltransferase. The relative positions of motifs (i) (SEQ IDNO:78), (ii) (SEQ ID NO:79) and (iii) (SEQ ID NO:80) align with residues231-243, 396-425 and 549-567, respectively, of the GTF 7527 sequence(SEQ ID NO:65) shown in FIGS. 2A-O. In certain embodiments herein,

-   -   (A) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:78 in the glucosyltransferase catalytic        domain aligns with amino acid positions 231-243 of SEQ ID NO:65;    -   (B) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:79 in the glucosyltransferase catalytic        domain aligns with amino acid positions 396-425 of SEQ ID NO:65;        and/or    -   (C) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:80 in the glucosyltransferase catalytic        domain aligns with amino acid positions 549-567 of SEQ ID NO:65.

The relative position(s) of the amino acid sequence(s) detected in theparent glucosyltransferase catalytic domain can thus be determined withreference to the above amino acid positions in SEQ ID NO:65. Forexample, the sequence of a putative parent glucosyltransferase catalyticdomain can be aligned with SEQ ID NO:65 using any means known in the artand/or as described above.

Alternatively, motif (i), (ii), and/or (iii) can be detected based onproximity to certain conserved sequences, namely SEQ ID NOs:72, 73, 74,75, 76 and 77, as described above.

In some embodiments, it is contemplated that detecting any one of motifs(i), (ii), or (iii) effectively results in identification of a parentglucosyltransferase catalytic domain having all three of these motifs.This being said, identifying a parent glucosyltransferase herein canoptionally comprise detecting one of, two of, or all three, of motifs(i), (ii) and/or (iii) in a glucosyltransferase catalytic domain.

A parent glucosyltransferase in a polynucleotide preparation methodherein can comprise a catalytic domain comprising an amino acid sequencethat is at least 90% identical to amino acid positions 54-957 of SEQ IDNO:65. Alternatively, a parent glucosyltransferase herein can comprise acatalytic domain having an amino acid sequence that is at least 90%identical to amino acid positions 55-960 of SEQ ID NO:30, positions55-960 of SEQ ID NO:4, positions 55-960 of SEQ ID NO:28, and/orpositions 55-960 of SEQ ID NO:20. Alternatively still, a parentglucosyltransferase catalytic domain can be detected that comprises anamino acid sequence that is 100% identical to, or at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, any ofthe foregoing sequences.

Certain of the conserved regions in FIGS. 2A-O include catalytic activesite motifs SEQ ID NOs:68, 69, 70, and 71 (refer to Example 3). Thus, acatalytic domain sequence of a parent glucosyltransferase enzyme in someaspects can be identified based on having one or more of SEQ ID NOs:68,69, 70, and 71 in alignment, respectively, with SEQ ID NOs:68, 69, 70,and 71 as present in amino acids 54-957 of SEQ ID NO:65. Other conservedregions in FIGS. 2A-O include SEQ ID NOs:72, 73, 74, 75, 76 and 77(refer to Example 4). Thus, a catalytic domain sequence of a parentglucosyltransferase enzyme in some aspects can be identified based onhaving one or more of SEQ ID NOs:72, 73, 74, 75, 76 and 77 in alignment,respectively, with SEQ ID NOs:72, 73, 74, 75, 76 and 77 as present inamino acids 54-957 of SEQ ID NO:65.

Although it is believed that a glucosyltransferase enzyme herein needonly have a catalytic domain sequence comprising an amino acid sequencethat is at least 90% identical to amino acid positions 54-957 of SEQ IDNO:65 (or positions 55-960 of SEQ ID NO:30, positions 55-960 of SEQ IDNO:4, positions 55-960 of SEQ ID NO:28, or positions 55-960 of SEQ IDNO:20), a parent glucosyltransferase enzyme identified in apolynucleotide preparation method herein is typically comprised within alarger amino acid sequence. For example, the catalytic domain may belinked at its C-terminus to a glucan-binding domain, and/or linked atits N-terminus to a variable domain and/or signal peptide.

The catalytic domain of a parent glucosyltransferase enzyme identifiedherein can have activity as exhibited by a catalytic domain of aglucosyltransferase classified under the glycoside hydrolase family 70(GH70). Such a GH70 glucosyltransferase may be found in the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009), for example.

Still further examples of parent glucosyltransferase enzymes in apolynucleotide preparation method herein can be any as disclosed hereinand that include 1-300 (or any integer there between [e.g., 10, 15, 20,25, 30, 35, 40, 45, or 50]) residues on the N-terminus and/orC-terminus. Such additional residues may be from a corresponding wildtype sequence from which the glucosyltransferase enzyme is derived, ormay be a heterologous sequence such as an epitope tag (at either N- orC-terminus) or a heterologous signal peptide (at N-terminus), forexample. Examples of such parent glucosyltransferase enzymes comprise anamino acid sequence that is 100% identical to, or at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to,SEQ ID NO:65, 30, 4, 28, or 20. These sequences (SEQ ID NO:65, 30, 4,28, 20) lack an N-terminal signal peptide (as well as a variable domain)(refer to Table 1). Still other examples of parent glucosyltransferaseenzymes herein include those comprising an amino acid sequence that is100% identical to, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 98.5%, 99%, or 99.5% A identical to, SEQ ID NO:60, 61, 62, 63, or64.

A parent glucosyltransferase identified in a polynucleotide preparationmethod herein can, for instance, synthesize insoluble polyalpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages andDP_(w) of at least 100. In certain embodiments, a parentglucosyltransferase enzyme can synthesize poly alpha-1,3-glucan in whichat least about 95%, 96%, 97%, 98%, 99%, or 100% of the constituentglycosidic linkages are alpha-1,3 linkages. In such embodiments,accordingly, the glucosyltransferase enzyme synthesizes polyalpha-1,3-glucan in which there is less than about 5%, 4%, 3%, 2%, 1%,or 0% of glycosidic linkages that are not alpha-1,3.

In another aspect, a parent glucosyltransferase enzyme can synthesizepoly alpha-1,3-glucan having no branch points or less than about 5%, 4%,3%, 2%, or 1% branch points as a percent of the glycosidic linkages inthe polymer. Examples of branch points include alpha-1,6 branch points.

In still another aspect, a parent glucosyltransferase enzyme cansynthesize poly alpha-1,3-glucan having a molecular weight in DP_(w) orDP_(n) of at least about 100. Alternatively, a parentglucosyltransferase enzyme may synthesize poly alpha-1,3-glucan having amolecular weight in DP_(n) or DP_(w) of at least about 400.Alternatively still, a parent glucosyltransferase enzyme may synthesizepoly alpha-1,3-glucan having a molecular weight in DP_(n) or DP_(w) ofat least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, or 1000 (or any integer between 100and 1000).

A method of preparing a polynucleotide sequence encoding aglucosyltransferase that produces a branched alpha-glucan polymercomprises step (b) of modifying the polynucleotide sequence (encoding aparent glucosyltransferase) identified in step (a). Such modificationdeletes and/or mutates (removes) at least one of motifs (i), (ii), or(iii) encoded by the polynucleotide sequence.

Modification of sequence encoding motif (i), (ii) and/or (iii) hereinallows expression of a child glucosyltransferase with a catalytic domainthat does not comprise amino acid sequence(s) that is/are at least 90%identical to SEQ ID NO:78 (motif i), SEQ ID NO:79 (motif ii), and/or SEQID NO:80 (motif iii). In some embodiments, a child glucosyltransferasecomprises a catalytic domain that does not comprise amino acidsequence(s) that is/are at least 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%,68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%,54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, or40% identical to SEQ ID NO:78, SEQ ID NO:79, and/or SEQ ID NO:80. Sincea parent glucosyltransferase can comprise a catalytic domain that is atleast 90% identical to positions 54-957 of SEQ ID NO:65, a childglucosyltransferase typically comprises a catalytic domain that has anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to positions 54-941 of SEQ ID NO:85,54-927 of SEQ ID NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQ ID NO:91,54-919 of SEQ ID NO:93, 54-905 of SEQ ID NO:95, or 54-889 of SEQ IDNO:97 (each of these sequences comprises one or more deleted motifscompared to positions 54-957 of SEQ ID NO:65).

A deletion or mutation can be directed to motif (i) only, motif (ii)only, motif (iii) only, both motifs (i) and (ii), both motifs (i) and(iii), both motifs (ii) and (iii), and all three of motifs (i), (ii) and(iii), for example. In certain embodiments, modification step (b)comprises deleting at least one of motifs (i), (ii), or (iii) encoded bythe polynucleotide sequence identified in step (a). Such deletion cancomprise removing most of (e.g., more than 70%, 80%, or 90% of), or alloff, one or more sequences encoding motif (i), (ii), or (iii).

If motif (i) is deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-941 of SEQ IDNO:85, for example.

If motif (ii) is deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-927 of SEQ IDNO:87, for example.

If motif (iii) is deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-935 of SEQ IDNO:89, for example.

If motifs (i) and (ii) are deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-911 of SEQ IDNO:91, for example.

If motifs (i) and (iii) are deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-919 of SEQ IDNO:93, for example.

If motifs (ii) and (iii) are deleted or mutated, then the encoded childglucosyltransferase can have a catalytic domain comprising an amino acidsequence that is at least 90% identical to positions 54-905 of SEQ IDNO:95, for example.

If motifs (i), (ii) and (iii) are deleted or mutated, then the encodedchild glucosyltransferase can have a catalytic domain comprising anamino acid sequence that is at least 90% identical to positions 54-889of SEQ ID NO:97, for example.

A deletion or mutation of a polynucleotide in modification step (b) canbe made following any DNA manipulation technique known in the art.Modifying step (b) can optionally be performed in silico, followed bysynthesis of the polynucleotide sequence encoding a glucosyltransferaseenzyme that produces a branched alpha-glucan polymer. For example, anucleotide sequence identified in step (a) can be manipulated in silicousing a suitable sequence manipulation program/software (e.g., VECTORNTI, Life Technologies, Carlsbad, Calif.; DNAStrider; DNASTAR, Madison,Wis.). Following such virtual manipulation, the modified polynucleotidesequence can be artificially synthesized by any suitable technique(e.g., annealing-based connection of oligonucleotides, or any techniquedisclosed in Hughes et al., Methods Enzymol. 498:277-309, which isincorporated herein by reference). It should be appreciated that theforegoing methodology is not believed to rely on having a pre-existingpolynucleotide sequence in hand.

Alternatively, modifying step (b) can optionally be performed using aphysical copy of a polynucleotide sequence identified in step (a)encoding a parent glucosyltransferase. As an example, such apolynucleotide can serve as a template for amplification using primersdesigned in a manner such that the amplified product has one or moredeletions (e.g., refer to Innis et al., above).

Suitable types of mutations that can be applied in step (b) in someaspects herein include those resulting in an amino acid substitution.One or more substitutions typically are non-conservative amino acidchanges.

A glucosyltransferase encoded by the polynucleotide sequence produced instep (b) (i.e., child glucosyltransferase) can produce branchedalpha-glucan. In some embodiments, alpha-glucan polymer branching can begauged using measurements of intrinsic viscosity and/or branching index(g′), as described above and in the below Examples.

Alpha-glucan polymer branching herein can, in some aspects, be judgedwith respect to measurements made against poly alpha-1,3-glucancontaining at least 95%, 96%, 97%, 98%, or 99% alpha-1,3 glycosidiclinkages (such polymer is expected to be mostly unbranched), or 100%alpha-1,3 glycosidic linkages (such polymer is linear/unbranched).Measurements can be with respect to intrinsic viscosity and/or branchingindex, for example. In certain embodiments, alpha-glucan produced by achild glucosyltransferase herein can have an intrinsic viscosity and/orbranching index (each measurement per methodology disclosed in belowExamples, for example) that is reduced by at least about 30%, 40%, 50%,60%, 70%, 80%, or 90% compared to poly alpha-1,3-glucan synthesized by aparent glucosyltransferase identified in step (a).

A branched alpha-glucan polymer produced by a child glucosyltransferaseherein is believed to contain at least both alpha-1,3 and alpha-1,6glycosidic linkages, for example. A branched alpha-glucan polymer maypossibly further comprise alpha-1,2 and/or alpha-1,4 glycosidic linkagesin some aspects. There are likely no beta-glycosidic linkages present.In certain embodiments, branched alpha-glucan polymer can have less than94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, or 30% alpha-1,3 glycosidic linkages. A branched alpha-glucanpolymer can have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%alpha-1,6 glycosidic linkages in some aspects. It is contemplated that,in some aspects, a branch point occurs on average about every 5, 10, 15,20, 25, 30, 35, or 40 monosaccharide units in a branched alpha-glucanherein.

A branched alpha-glucan polymer in most embodiments is insoluble. Suchinsolubility is observed in aqueous conditions (e.g., solvent comprisingat least 90% water) of generally neutral pH (e.g., between 6-8), forexample.

Some embodiments disclosed herein concern a polynucleotide sequenceproduced following the above method of preparing a polynucleotidesequence. Such a polynucleotide sequence encodes a glucosyltransferasethat produces a branched alpha-glucan polymer. Optionally, one or moreregulatory sequences are operably linked to the nucleotide sequence, andpreferably a promoter sequence is included as a regulatory sequence.Additional possible features of a polynucleotide sequence are describedabove.

Still other aspects disclosed herein concern a glucosyltransferase(child glucosyltransferase) encoded by such a polynucleotide sequence.Features of such a glucosyltransferase can be any as disclosed above.

Some other embodiments of the present disclosure are drawn to branchedalpha-glucan polymer produced by a glucosyltransferase herein (e.g., achild glucosyltransferase herein; a glucosyltransferase comprising SEQID NO:85, 87, 89, 91, 93, 95, or 97).

In other embodiments, reaction solutions are disclosed that comprisewater, sucrose, and one or more glucosyltransferase enzymes herein thatproduce a branched alpha-glucan polymer. Other components can optionallybe comprised within a reaction solution for synthesizing branchedalpha-glucan, such as fructose, glucose, leucrose, and solubleoligosaccharides (e.g., DP2-DP7). It would be understood that certainbranched alpha-glucan products herein may be water-insoluble and thusnot dissolved in a glucan synthesis reaction, but rather may be presentout of solution. A reaction solution herein may be one that, in additionto producing insoluble glucan product, produces byproducts such asleucrose and/or soluble oligosaccharides.

The temperature of a reaction solution herein can be controlled, ifdesired. In certain embodiments, the temperature of the reaction can bebetween about 5° C. to about 50° C. The temperature in certain otherembodiments can be between about 20° C. to about 40° C., or about 20° C.to about 30° C. (e.g., about 22-25° C.).

The initial concentration of sucrose in a reaction solution herein canbe about 20 g/L to about 400 g/L, for example. Alternatively, theinitial concentration of sucrose can be about 75 g/L to about 175 g/L,or from about 50 g/L to about 150 g/L. Alternatively still, the initialconcentration of sucrose can be about 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, or 160 g/L (or any integer value between 40 and 160g/L), for example. “Initial concentration of sucrose” refers to thesucrose concentration in a GTF reaction solution just after all thereaction solution components have been added (e.g., at least water,sucrose, GTF enzyme).

Sucrose used in a glucan synthesis reaction herein can be highly pure(≥99.5%) or be of any other purity or grade. For example, sucrose canhave a purity of at least 99.0%, or can be reagent grade sucrose. Asanother example, incompletely refined sucrose can be used. Incompletelyrefined sucrose herein refers to sucrose that has not been processed towhite refined sucrose. Thus, incompletely refined sucrose can becompletely unrefined or partially refined. Examples of unrefined sucroseare “raw sucrose” (“raw sugar”) and solutions thereof. Examples ofpartially refined sucrose have not gone through one, two, three, or morecrystallization steps. The ICUMSA (International Commission for UniformMethods of Sugar Analysis) of incompletely refined sucrose herein can begreater than 150, for example. Sucrose herein may be derived from anyrenewable sugar source such as sugar cane, sugar beets, cassava, sweetsorghum, or corn. Suitable forms of sucrose useful herein arecrystalline form or non-crystalline form (e.g., syrup, cane juice, beetjuice), for example.

Methods of determining ICUMSA values for sucrose are well known in theart and disclosed by the International Commission for Uniform Methods ofSugar Analysis in ICUMSA Methods of Sugar Analysis: Official andTentative Methods Recommended by the International Commission forUniform Methods of Sugar Analysis (ICUMSA) (Ed. H. C. S. de Whalley,Elsevier Pub. Co., 1964), for example, which is incorporated herein byreference. ICUMSA can be measured, for example, by ICUMSA Method GS1/3-7as described by R. J. McCowage, R. M. Urquhart and M. L. Burge(Determination of the Solution Colour of Raw Sugars, Brown Sugars andColoured Syrups at pH 7.0—Official, Verlag Dr Albert Bartens, 2011revision), which is incorporated herein by reference.

The pH of a glucan synthesis reaction in certain embodiments can bebetween about 4.0 to about 8.0. Alternatively, the pH can be about 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The pH can be adjusted orcontrolled by the addition or incorporation of a suitable buffer,including but not limited to: phosphate, tris, citrate, or a combinationthereof. Buffer concentration in a glucan synthesis reaction can be from0 mM to about 100 mM, or about 10, 20, or 50 mM, for example.

One or more different glucosyltransferase enzymes that produce branchedalpha-glucan may be used in certain aspects. A reaction solution hereinmay contain one, two, or more glucosyltransferase enzymes, for example.

The present disclosure also concerns a method for producing branchedalpha-glucan polymer, the method comprising:

(a) contacting at least water, sucrose, and one or moreglucosyltransferase enzymes as disclosed herein that produce branchedalpha-glucan polymer, whereby branched alpha-glucan polymer is produced,and

b) optionally, isolating the alpha-glucan polymer produced in step (a).

A glucan synthesis method as presently disclosed comprises contacting atleast water, sucrose, and a glucosyltransferase enzyme as describedherein that synthesizes branched alpha-glucan. These and optionallyother reagents can be added altogether or added in any order asdiscussed below. This step can comprise providing a reaction solutioncomprising water, sucrose and a glucosyltransferase enzyme thatsynthesizes branched alpha-glucan. In certain embodiments in whichinsoluble branched alpha-glucan is synthesized by a glucosyltransferase,it would be understood that the reaction solution becomes a reactionmixture given that insoluble glucan polymer falls out of solution. Thecontacting step herein can be performed in any number of ways. Forexample, the desired amount of sucrose can first be dissolved in water(optionally, other components may also be added at this stage ofpreparation, such as buffer components), followed by addition ofglucosyltransferase enzyme. The solution may be kept still, or agitatedvia stirring or orbital shaking, for example. Typically, a glucansynthesis reaction is cell-free.

Completion of a reaction in certain embodiments can be determinedvisually (e.g., no more accumulation of insoluble glucan) and/or bymeasuring the amount of sucrose left in the solution (residual sucrose),where a percent sucrose consumption of over about 90% can indicatereaction completion, for example. Typically, a reaction of the disclosedprocess will take about 12, 24, 36, 48, 60, 72, 84, or 96 hours tocomplete, depending on certain parameters such as the amount of sucroseand glucosyltransferase enzyme used in the reaction.

The yield of branched alpha-glucan produced in some aspects of a glucansynthesis method herein can be at least about 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, based on the weightof sucrose converted in the reaction.

Branched alpha-glucan produced in the disclosed method may optionally beisolated. For example, insoluble branched alpha-glucan may be separatedby centrifugation or filtration. In doing so, the glucan is separatedfrom most of the reaction solution, which may comprise water, fructoseand certain byproducts (e.g., leucrose, soluble oligosaccharidesDP2-DP7). This solution may also comprise residual sucrose and glucosemonomer. Isolation can optionally further comprise washing branchedglucan product one, two, or more times with water or other aqueousliquid, and/or drying the glucan product.

The above embodiments of branched alpha-glucan synthesis methods areexamples. Any other feature disclosed herein can apply to a branchedglucan synthesis method, accordingly. For example, any of the branchedglucan product, glucosyltransferase enzyme (e.g., the catalytic domainand its motif profile), and reaction solution condition featuresdisclosed herein can be applied as appropriate.

Non-limiting examples of compositions and methods disclosed hereininclude:

-   1. A glucosyltransferase enzyme comprising a catalytic domain that    comprises an amino acid sequence that is at least 90% identical to    amino acid positions: 54-941 of SEQ ID NO:85, 54-927 of SEQ ID    NO:87, 54-935 of SEQ ID NO:89, 54-911 of SEQ ID NO:91, 54-919 of SEQ    ID NO:93, 54-905 of SEQ ID NO:95, or 54-889 of SEQ ID NO:97, wherein    the catalytic domain lacks at least one motif selected from the    group consisting of:    -   (i) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:78,    -   (ii) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:79, and    -   (iii) a motif comprising an amino acid sequence that is at least        90% identical to SEQ ID NO:80;    -   wherein the glucosyltransferase enzyme produces a branched        alpha-glucan polymer.-   2. The glucosyltransferase of embodiment 1, wherein the    glucosyltransferase comprises an amino acid sequence that is at    least 90% identical to SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ    ID NO:91, SEQ ID NO:93, SEQ ID NO:95, or SEQ ID NO:97, and wherein    the glucosyltransferase lacks at least one of motifs (i), (ii), or    (iii).-   3. A polynucleotide comprising a nucleotide sequence encoding a    glucosyltransferase enzyme according to embodiment 1 or 2,    optionally wherein one or more regulatory sequences are operably    linked to the nucleotide sequence, and preferably wherein the one or    more regulatory sequences include a promoter sequence.-   4. A method of preparing a polynucleotide sequence encoding a    glucosyltransferase enzyme that produces a branched alpha-glucan    polymer, the method comprising:    -   (a) identifying a polynucleotide sequence encoding a parent        glucosyltransferase enzyme that comprises a catalytic domain        comprising:        -   (1) an amino acid sequence that is at least 90% identical to            amino acid positions 54-957 of SEQ ID NO:65, and        -   (2) the following three motifs:            -   (i) a motif comprising an amino acid sequence that is at                least 90% identical to SEQ ID NO:78,            -   (ii) a motif comprising an amino acid sequence that is                at least 90% identical to SEQ ID NO:79, and            -   (iii) a motif comprising an amino acid sequence that is                at least 90% identical to SEQ ID NO:80;            -   and    -   (b) modifying the polynucleotide sequence identified in step (a)        to delete and/or mutate at least one of motifs (i), (ii),        or (iii) encoded by the polynucleotide sequence, thereby        providing a polynucleotide sequence encoding a        glucosyltransferase enzyme that produces a branched alpha-glucan        polymer.-   5. The method of embodiment 4, wherein:    -   (A) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:78 aligns with amino acid positions        231-243 of SEQ ID NO:65;    -   (B) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:79 aligns with amino acid positions        396-425 of SEQ ID NO:65; and/or    -   (C) the position of the amino acid sequence that is at least 90%        identical to SEQ ID NO:80 aligns with amino acid positions        549-567 of SEQ ID NO:65.-   6. The method of embodiment 4 or 5, wherein motif (i) comprises SEQ    ID NO:78, motif (ii) comprises SEQ ID NO:79, and motif (iii)    comprises SEQ ID NO:80.-   7. The method of embodiment 4, 5, or 6, wherein the parent    glucosyltransferase enzyme can synthesize poly alpha-1,3-glucan    having at least 95% alpha-1,3 glycosidic linkages and a weight    average degree of polymerization (DP_(w)) of at least 100.-   8. The method of embodiment 4, 5, 6, or 7, wherein modification    step (b) comprises deleting at least one of motifs (i), (ii),    or (iii) encoded by the polynucleotide sequence identified in step    (a).-   9. The method of embodiment 4, 5, 6, 7, or 8, wherein the    glucosyltransferase enzyme of step (b) comprises a catalytic domain    that does not comprise at least one amino acid sequence that is at    least 60% identical to SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80.-   10. The method of embodiment 4, 5, 6, 7, 8, or 9, wherein the    branched alpha-glucan polymer has an intrinsic viscosity and/or    branching index that is reduced by at least 30% compared to the    intrinsic viscosity and/or branching index of poly alpha-1,3-glucan    synthesized by the parent glucosyltransferase.-   11. The method of embodiment 4, 5, 6, 7, 8, 9, or 10,    -   wherein the identifying step is performed:        -   (a) in silico,        -   (b) with a method comprising a nucleic acid hybridization            step,        -   (c) with a method comprising a protein sequencing step,            and/or        -   (d) with a method comprising a protein binding step;    -   and/or wherein the modifying step is performed:        -   (e) in silico, followed by synthesis of the polynucleotide            sequence encoding the glucosyltransferase enzyme that            produces a branched alpha-glucan polymer, or        -   (f) using a physical copy of the polynucleotide sequence            encoding the parent glucosyltransferase.-   12. A polynucleotide sequence encoding a glucosyltransferase enzyme    that produces a branched alpha-glucan polymer, wherein the    polynucleotide sequence is produced according to the method of    embodiment 4, 5, 6, 7, 8, 9, 10, or 11, optionally wherein the    polynucleotide sequence further comprises one or more regulatory    sequences operably linked to the polynucleotide sequence, preferably    wherein the one or more regulatory sequences include a promoter    sequence.-   13. A glucosyltransferase enzyme encoded by the polynucleotide of    embodiment 12.-   14. A reaction solution comprising water, sucrose, and a    glucosyltransferase enzyme according to embodiment 1, 2, or 13.-   15. A method for producing a branched alpha-glucan polymer    comprising:    -   (a) contacting at least water, sucrose, and a        glucosyltransferase enzyme according to embodiment 1, 2, or 13,        whereby branched alpha-glucan polymer is produced, and    -   b) optionally, isolating the branched alpha-glucan polymer        produced in step (a).-   16. A branched alpha-glucan polymer, wherein the polymer is produced    from a method according to embodiment 15 or from a reaction solution    according to embodiment 14, or wherein the polymer is a product of a    glucosyltransferase according to any of embodiments 1-2.

EXAMPLES

The present disclosure is further exemplified in the following Examples.It should be understood that these Examples, while indicating certainpreferred aspects herein, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the disclosed embodiments,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt the disclosed embodiments tovarious uses and conditions.

Abbreviations

The meanings of some of the abbreviations used herein are as follows:“g” means gram(s), “h” means hour(s), “mL” means milliliter(s), “psi”means pound(s) per square inch, “wt %” means weight percentage, “μm”means micrometer(s), “° C.” means degrees Celsius, “mg” meansmilligram(s), “mm” means millimeter(s), “μL” means microliter(s), “mmol”means millimole(s), “min” means minute(s), “mol %” means mole percent,“M” means molar, “rpm” means revolutions per minute, “MPa” meansmegaPascals, “IV” means intrinsic viscosity, “g” means branching ratio.

General Methods

Preparation of Crude Extracts of Glucosyltransferase (GTF) Enzymes

GTF enzymes were prepared as follows. E. coli TOP10® cells (Invitrogen,Carlsbad, Calif.) were transformed with a pJexpress404®-based constructcontaining a particular GTF-encoding DNA sequence. Each sequence wascodon-optimized to express the GTF enzyme in E. coli. Individual E. colistrains expressing a particular GTF enzyme were grown in LB (Luriabroth) medium (Becton, Dickinson and Company, Franklin Lakes, N.J.) withampicillin (100 μg/mL) at 37° C. with shaking to OD₆₀₀=0.4-0.5, at whichtime IPTG (isopropyl beta-D-1-thiogalactopyranoside, Cat. No. 16758,Sigma-Aldrich, St. Louis, Mo.) was added to a final concentration of 0.5mM. The cultures were incubated for 2-4 hours at 37° C. following IPTGinduction. Cells were harvested by centrifugation at 5,000×g for 15minutes and resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0supplemented with dithiothreitol (DTT, 1.0 mM). Resuspended cells werepassed through a French Pressure Cell (SLM Instruments, Rochester, N.Y.)twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30minutes at 12,000 x g at 4° C. The resulting supernatant was analyzed bythe BCA (bicinchoninic acid) protein assay (Sigma-Aldrich) and SDS-PAGEto confirm expression of the GTF enzyme, and the supernatant was storedat −20° C.

Determination of GTF Enzymatic Activity

GTF enzyme activity was confirmed by measuring the production ofreducing sugars (fructose and glucose) in a GTF reaction solution. Areaction solution was prepared by adding a GTF extract (prepared asabove) to a mixture containing sucrose (50 or 150 g/L), potassiumphosphate buffer (pH 6.5, 50 mM), and optionally dextran (1 mg/mL,dextran T10, Cat. No. D9260, Sigma-Aldrich); the GTF extract was addedto 2.5%-5% by volume. The reaction solution was then incubated at 22-25°C. for 24-30 hours, after which it was centrifuged. Supernatant (0.01mL) was added to a mixture containing 1 N NaOH and 0.1%triphenyltetrazolium chloride (Sigma-Aldrich). The mixture was incubatedfor five minutes after which its OD₄₈₀ was determined using an ULTROSPECspectrophotometer (Pharmacia LKB, New York, N.Y.) to gauge the presenceof the reducing sugars fructose and glucose.

Determination of Glycosidic Linkages

Glycosidic linkages in the glucan product synthesized by a GTF enzymewere determined by ¹³C NMR (nuclear magnetic resonance). Dry glucanpolymer (25-30 mg) was dissolved in 1 mL of deuterated dimethylsulfoxide (DMSO) containing 3% by weight of LiCl with stirring at 50° C.Using a glass pipet, 0.8 mL of the solution was transferred into a 5-mmNMR tube. A quantitative ¹³C NMR spectrum was acquired using a BrukerAvance 500-MHz NMR spectrometer (Billerica, Mass.) equipped with a CPDULcryoprobe at a spectral frequency of 125.76 MHz, using a spectral windowof 26041.7 Hz. An inverse gated decoupling pulse sequence using waltzdecoupling was used with an acquisition time of 0.629 second, aninter-pulse delay of 5 seconds, and 6000 pulses. The time domain datawas transformed using an exponential multiplication of 2.0 Hz.

Determination of Number Average Degree of Polymerization DP_(n))

The DP_(n) of a glucan product synthesized by a GTF enzyme wasdetermined by size-exclusion chromatography (SEC). Dry glucan polymerwas dissolved at 5 mg/mL in N,N-dimethyl-acetamide (DMAc) and 5% LiClwith overnight shaking at 100° C. The SEC system used was an Alliance™2695 separation module from Waters Corporation (Milford, Mass.) coupledwith three on-line detectors: a differential refractometer 2410 fromWaters, a multiangle light scattering photometer Heleos™ 8+ from WyattTechnologies (Santa Barbara, Calif.), and a differential capillaryviscometer ViscoStar™ from Wyatt. The columns used for SEC were fourstyrene-divinyl benzene columns from Shodex (Japan) and two linearKD-806M, KD-802 and KD-801 columns to improve resolution at the lowmolecular weight region of a polymer distribution. The mobile phase wasDMAc with 0.11% LiCl. The chromatographic conditions used were 50° C. inthe column and detector compartments, 40° C. in the sample and injectorcompartment, a flow rate of 0.5 mL/min, and an injection volume of 100μL. The software packages used for data reduction were Empower™ version3 from Waters (calibration with broad glucan polymer standard) andAstra® version 6 from Wyatt (triple detection method with columncalibration).

Determination of Intrinsic Viscosity

Multidetector size exclusion chromatography (SEC) allowed measurement ofmolar mass distribution (MMD) using a combination of light scattering(LS) photometer and differential refractometer (DR). Molar mass (M) ofthe separated fractions across the polymer distribution was measured asa ratio of two detector responses:

M˜LS/DR, without any column calibration.

In a similar way, an in-line differential viscometer (DV) allowedmeasurement of intrinsic viscosity (IV) of the separated fractions:

IV˜DV/DR.

By plotting IV as a function of M in log-log scale, a so-calledMark-Houwink plot was obtained for samples tested.

Determination of Branching Ratio

Mark-Houwink (MH) plots were useful for estimating the degree ofbranching in polymers through measuring their size as a function ofmolar mass. Thus, the hydrodynamic size (H) of the macromolecule indilute solution was determined as H=IV×M, so that using an MH plot, itcould be seen how the size of the polymer chain changes with its molarmass. Branched polymer has a smaller size in solution than its linearcounterpart with the same molar mass, and the position of the MH-plotindicates the degree of polymer branching.

To quantify the degree of branching, the branching ratio (or branchingindex) g′ was plotted as a function of molar mass. This index is definedas a ratio of hydrodynamic volume of branched polymer chain H_(br) witha given molar mass M, to the similar volume H_(lin) of the linear chainwith the same molar mass; i.e., g′(M)=H_(br)/H_(lin). Since H is definedas a production of IV and M, and M is the same in both numerator anddenominator, then g′ could be determined for each separated fractionwith molar mass M directly from the corresponding MH plots asg′=IV_(br)/IV_(lin). These plots show how the degree of branchingchanges with the polymer molar mass. The weight-average branching indexfor each polymer (i.e., g′=IV_(br,w)/IV_(lin,w)) was a useful estimationof the overall branching frequency in the polydispersed polymer. A g′value of 1, per this analysis, indicates that a polymer is linear(unbranched), whereas a g′ value<1 indicates that a polymer is branched.

Example 1 Production of GTF Enzymes

This Example describes the preparation of N-terminally truncatedversions of glucosyltransferase (GTF) enzymes used in this study.

Nucleotide sequences encoding N-terminally truncated versions of GTFenzymes (Table 2, GTF ID) were synthesized using codons optimized forprotein expression in E. coli. The nucleic acid products (Table 2, ntSEQ ID NO) encoding the GTF enzymes (Table 2, AA SEQ ID NO) weresubcloned into pJexpresss404® (DNA2.0, Menlo Park, Calif.) to generateGTF expression plasmids (Table 2, plasmid ID). The GTF expressionplasmids were used to transform E. coli TOP10 cells (Invitrogen,Carlsbad, Calif.) to generate GTF expression strains (Table 2, strainID). Production of GTF enzymes by bacterial expression and determinationof enzymatic activities were performed as described in General Methods.

TABLE 2 Production of GTF Enzymes AA nt SEQ SEQ ID Plasmid GTF ID GINo.^(a) ID NO NO ID Strain ID 0874 450874 1 2 pMP53 TOP10/pMP53 6855228476855 3 4 pMP66 TOP10/pMP66 2379 662379 5 6 pMP65 TOP10/pMP65 752747527 7 8 pMP52 TOP10/pMP52 1724 121724 9 10 pMP55 TOP10/pMP55 0544290580544 11 12 pMP67 TOP10/pMP67 5926 167735926 13 14 pMP56 TOP10/pMP564297 7684297 15 16 pMP70 TOP10/pMP70 5618 328945618 17 18 pMP72TOP10/pMP72 2765 322372765 19 20 pMP85 TOP10/pMP85 4700 21654700 21 22pMP83 TOP10/pMP83 1366 146741366 23 24 pMP86 TOP10/pMP86 0427 940427 2526 pMP87 TOP10/pMP87 2919 383282919 27 28 pMP88 TOP10/pMP88 2678400182678 29 30 pMP89 TOP10/pMP89 2381 662381 31 32 pMP96 TOP10/pMP963929 387783929 33 34 pMP97 TOP10/pMP97 6907 228476907 35 36 pMP57TOP10/pMP57 6661 228476661 37 38 pMP62 TOP10/pMP62 0339 334280339 39 40pMP73 TOP10/pMP73 0088 3130088 41 42 pMP69 TOP10/pMP69 9358 24379358 4344 pMP71 TOP10/pMP71 8242 325978242 45 46 pMP68 TOP10/pMP68 3442324993442 47 48 pMP75 TOP10/pMP75 7528 47528 49 50 pMP77 TOP10/pMP773279 322373279 51 52 pMP79 TOP10/pMP79 6491 170016491 53 54 pMP74TOP10/pMP74 6889 228476889 55 56 pMP60 TOP10/pMP60 4154 51574154 57 58pMP80 TOP10/pMP80 3298 322373298 59 pMP98 TOP10/pMP98 ^(a)GI number asprovided for each respective sequence in GENBANK database (NCBI).

Example 2 Production of Glucan Polymer using GTF Enzymes

This Example describes using the GTF enzymes prepared in Example 1 tosynthesize glucan polymer.

Polymerization reactions were performed with each of the GTF enzymesprepared in Example 1. Reaction solutions were prepared comprisingsucrose (50 g/L), potassium phosphate buffer (pH 6.5, 20 mM) and a GTFenzyme (2.5% extract by volume). After 24-30 hours at 22-25° C.,insoluble glucan polymer product was harvested by centrifugation, washedthree times with water, washed once with ethanol, and dried at 50° C.for 24-30 hours.

Glycosidic linkages in each insoluble glucan polymer product weredetermined by ¹³C NMR, and the DP_(n) for each insoluble polymer productwas determined by SEC, as described in General Methods. Thesemeasurements are provided in Table 3 below.

TABLE 3 Polymer produced by GTF enzymes Glucan Polymer SEQ ID ReducingInsoluble Linkages GTF ID NO. Sugars Product %1.3 %1.6 DP_(n) 0874 2 yesyes 100 0 60 6855 4 yes yes 100 0 440 2379 6 yes yes 37 63 310 7527 8yes yes 100 0 440 1724 10 yes yes 100 0 250 0544 12 yes yes 62 36 9805926 14 yes yes 100 0 260 4297 16 yes yes 31 67 800 5618 18 yes yes 3466 1020 2765 20 yes yes 100 0 280 4700 22 yes no 1366 24 yes no 0427 26yes yes 100 0 120 2919 28 yes yes 100 0 250 2678 30 yes yes 100 0 3902381 32 yes no 3929 34 yes yes 100 0 280 6907 36 yes no 6661 38 yes no0339 40 yes no 0088 42 yes no 9358 44 yes no 8242 46 yes no 3442 48 yesno 7528 50 yes no 3279 52 yes no 6491 54 yes no 6889 56 yes no 4154 58yes no 3298 59 yes no 50 50 none na no no

The following GTF enzymes produced glucan polymers comprising at least50% alpha-1,3-linkages and having a DP_(n) of at least 100: 6855 (SEQ IDNO:4), 7527 (SEQ ID NO:8), 1724 (SEQ ID NO:10), 0544 (SEQ ID NO:12),5926 (SEQ ID NO:14), 2765 (SEQ ID NO:20), 0427 (SEQ ID NO:26), 2919 (SEQID NO:28), 2678 (SEQ ID NO:30), and 3929 (SEQ ID NO:34) (refer to Table3). The following GTF enzymes produced glucan polymers comprising 100%alpha-1,3-linkages, indicating linear polymers: 6855 (SEQ ID NO:4), 7527(SEQ ID NO:8), 1724 (SEQ ID NO:10), 5926 (SEQ ID NO:14), 2765 (SEQ IDNO:20), 0427 (SEQ ID NO:26), 2919 (SEQ ID NO:28), 2678 (SEQ ID NO:30),and 3929 (SEQ ID NO:34). These results clearly indicate that not all GTFenzymes are capable of producing linear alpha-1,3-glucan polymer.

Example 3 Structure/Function Relationships Observed in GTF Sequences

This Example describes aligning the amino acid sequences of several GTFenzymes to determine whether they share any structures.

GTF enzymes were evaluated in Example 2 for their ability to produceglucan polymers with a focus on those enzymes that produce glucan with100% alpha-1,3-linkages. The sequences of several of these enzymes werealigned with three dimensional structures that are formed by certain S.mutans and L. reuteri GTF sequences (3AIE [SEQ ID NO:66] and 3KLK [SEQID NO:67], respectively); the S. mutans and L. reuteri GTF sequenceswere aligned to superpose common tertiary structures using the softwarepackage MOE (Chemical Computing Group, Montreal, Canada). The sequencesfor each of the GTF enzymes used in the alignment contain the catalyticand glucan-binding domains of each enzyme, respectively (i.e., theN-terminal signal peptide and variable domains of each GTF are notincluded in the alignment). FIGS. 2A-O show the alignment. The sequencesof the S. mutans and L. reuteri GTFs for which crystallographicstructures are known were included in the alignment; S. mutans GTF isabbreviated as “3AIE” (SEQ ID NO:66) and L. reuteri GTF is abbreviatedas “3KLK” (SEQ ID NO:67) in FIGS. 2A-O.

The alignment in FIGS. 2A-O indicates that all the aligned GTF sequencesmaintain numerous invariant regions (shown with dark background). Theseinvariant sequences are located throughout the catalytic domain of eachGTF (based on a homology model as opposed to an experimentallydetermined structure). The catalytic domains in the aligned GTFs areabout 900-950 amino acid residues long and begin after position 1(artificial start methionine) in each of the sequences shown in FIGS.2A-O. The sequence following the catalytic domain in each GTF representsthe glucan-binding domain. The aligned GTF sequences share as little as40% sequence identity with the sequences of the known GTF structures (S.mutans 3AIE and L. reuteri 3KLK). But the alignment of these sequencesin FIGS. 2A-O indicates a distributed pattern of conserved sequencemotifs and patterns of specific residues that are conserved in all thealigned sequences (residues with dark background in FIGS. 2A-O). Theseconserved sequence motifs can be related to important structuralfeatures such as the catalytic site described below and can serve asreference points to identify unique or characteristic features that maybe associated with specific performance benefits.

The catalytic site residues may be found in sequence motifs repeated inall the aligned sequences (FIGS. 2A-O). Specifically, with reference tothe sequence from GTF 7527 (SEQ ID N0:65) in FIGS. 2A-O, Arg292 andAsp294 are found in the motif FDxxRxDAxDNV (SEQ ID N0:68) correspondingto Arg475 and Asp477 of S. mutans 3AIE GTF and Arg1023 and Asp1025 of L.reuteri 3KLK GTF; Glu332 is found in the sequence motif ExWxxxDxxY (SEQID N0:69) corresponding to Glu515 in S. mutans 3AIE GTF and Glu1063 inL. reuteri 3KLK GTF; His434 and Asp435 are found in the sequence motifFxRAHD (SEQ ID NO:70) corresponding to His587 and Asp588 in S. mutans3AIE GTF and His1135 and Asp1136 in L. reuteri 3KLK GTF; and Tyr(Y)783is found in the sequence motif IxNGYAF (SEQ ID N0:71) corresponding tothe residues Tyr916 of S. mutans 3AIE GTF and Tyr1465 of L. reuteri 3KLKGTF.

Thus, the tested GTF enzymes have catalytic domains comprising severalhighly conserved regions.

Example 4 Sequence Motifs in GTF Enzymes that Synthesize High MolecularWeight Alpha-1,3-Glucan

The GTF enzymes whose sequences were aligned in FIGS. 2A-O were furtherevaluated for their ability to produce glucan polymers with a focus onthose enzymes that produce glucan with 100% alpha-1,3-linkages (Table4).

TABLE 4 Polymer Produced by Various GTF Enzymes Glucan Polymer FeaturesSEQ % Alpha- Cat. % Cat. GTF ID 1,3 % Domain Domain ID NO. Linkages^(a)DP_(w)50^(b) DP_(w)150^(b) Identity^(d) Region^(e) Identity^(f) 7527^(c)65 100 910 577 100 54-957 100 2678 30 100 740 657 94.1 55-960 94.9 68554 100 835 570 98.9 55-960 99.0 2919 28 100 600 414 93.1 55-960 95.5 276520 100 670 93.6 55-960 96.4 0088 42 <30 44.7 55-900 50.4 0544 12 62 46.755-900 51.2 0427 26 100 260 43.1 55-900 51.8 0874 2 100 105 50 43.355-900 52.0 1724 10 100 535 55 42.9 55-900 51.3 5926 14 100 475 68 46.055-900 50.9 1366 24 <30 46.1 55-900 50.9 3298 59 <30 44.1 55-910 49.82379 6 37 44.5 60-915 50.7 6907 36 <30 55.6 55-885 61.8 5618 18 34 46.255-905 51.4 4297 16 31 46.5 55-905 51.2 3442 48 <30 45.8 55-905 51.09358 44 <30 49.7 55-915 53.6 6661 38 <30 45.6 55-895 50.5 0339 40 <3053.7 55-895 57.5 8242 46 <30 54.1 55-910 59.4 7528 50 <30 48.1 55-91554.2 3279 52 <30 41.8 55-900 48.7 ^(a)Glucan products having <30%alpha-1,3 linkages were soluble and not further analyzed for DP_(w).^(b)DP_(w)50 and DP_(w)150 represent, respectively, the DP_(w) of glucanproduced by a GTF in a reaction solution having an initial sucroseconcentration of 50 g/L or 150 g/L. ^(c)SEQ ID NO: 65 is a shorterversion of the 7527 GTF of SEQ ID NO: 8. ^(d)Percent identity ofrespective GTF with SEQ ID NO: 65 (per EMBOSS alignment). ^(e)Amino acidposition of region within catalytic domain sequence having conservation(FIGS. 2A-O) with other listed GTF sequences (approximate location).^(f)Percent identity of catalytic domain region with amino acid residues54-957 of SEQ ID NO: 65 (per EMBOSS alignment).

Nine of the aligned GTF enzymes were found to produce glucan with 100%alpha-1,3-linkages, and five of these nine enzymes produced highmolecular weight polymer (DP_(w)>400, Table 4). Specifically, the fiveGTF enzymes that displayed the property of producing high molecularweight glucan with 100% alpha-1,3-linkages are 7527 (SEQ ID NO:65), 2678(SEQ ID NO:30), 6855 (SEQ ID NO:4), 2919 (SEQ ID NO:28) and 2765 (SEQ IDNO:20). The sequences for each of these GTFs are indicated with a “++”in (FIGS. 2A-O).

Three sequence motifs were found in the amino acid sequences of all fiveGTF enzymes that produce high molecular weight glucan with 100%alpha-1,3-linkages, and appear as three different “insertions” situatedaround the catalytic domain of the known GTF structures. Briefly, thesesequence motifs are designated as:

Motif 1a (SEQ ID NO: 78): D/N-K-S-I/V-L-D-E-Q-S-D-P-N-HMotif 2 (SEQ ID NO: 79):N-K-D-G-S-K/T-A-Y-N-E-D-G-T-V/A-K-Q/K-S-T-I-G-K- Y-N-E-K-Y-G-D-A-SMotif 3a (SEQ ID NO: 80): L-P-T-D-G-K-M-D-N/K-S-D-V-E-L-Y-R-T-N/S-E

The relative positions of motifs 1a, 2 and 3a align with residues231-243, 396-425 and 549-567, respectively, of the 7527 GTF sequence(SEQ ID NO:65) in FIGS. 2A-O. These motifs appear to be conserved amongGTF enzymes that synthesize high molecular weight alpha-1,3-glucan.

In the alignment shown in FIGS. 2A-O, motif 1a is flanked by upstreamand downstream sequences as shown in FIG. 3. Specifically, precedingmotif 1a is the sequence SxxRxxN (SEQ ID NO:72), and following motif 1ais the sequence GGxxxLLxNDxDxSNPxVQAExLN (SEQ ID NO:73). Both of thesesequences were found in all the aligned GTF sequences and can serve asreference points for identifying motif 1a in other GTF sequences. In thealignment shown in FIGS. 2A-O, motif 2 is flanked by upstream anddownstream sequences as shown in FIG. 5. Specifically, preceding motif 2by about 50 amino acids is the sequence WxxxDxxY (SEQ ID NO:74) andfollowing motif 2 is the sequence YxFxRAHD (SEQ ID NO:75). Thedownstream sequence (SEQ ID NO:75) includes two of the active siteresidues, His587 and Asp588 (numbered with respect to the S. mutans GTFstructure, 3AIE). Both of these sequences were found in all the alignedGTF sequences and can serve as reference points for identifying motif 2in other GTF sequences. In the alignment shown in FIGS. 2A-O, motif 3ais flanked by upstream and downstream sequences as shown in FIG. 7.Specifically, preceding motif 3a is sequence YxxGGQ (SEQ ID NO:76) andfollowing motif 3a is the sequence VRxG (SEQ ID NO:77). Both of thesesequences were found in all the aligned GTF sequences and can serve asreference points for identifying motif 2 in other GTF sequences.

Identification of motifs 1a (SEQ ID NO:78), 2 (SEQ ID NO:79) and 3a (SEQID NO:80) in the catalytic domains of GTF enzymes that synthesize highmolecular weight glucan having 100% alpha-1,3-glycosidic linkagesindicates that each of these motifs may be useful for identifying otherGTFs with similar activity.

Example 5 Sequence Motifs in GTF Enzymes that Synthesize Low MolecularWeight Alpha-1,3-Glucan

Four GTF enzymes produced low molecular weight glucan having 100%alpha-1,3-linkages (Table 4). Specifically, these enzymes were 5926 (SEQID NO: 14), 0427 (SEQ ID NO: 26), 0874 (SEQ ID NO: 2) and 1724 (SEQ IDNO: 10). The sequences for each of these enzymes are indicated with a“+−” in FIGS. 2A-O. Two sequence motifs were found in the amino acidsequences of these GTF enzymes, and appear as two different “insertions”situated around the catalytic domain of the known GTF structures.Briefly, these sequence motifs are designated as:

Motif 1b (SEQ ID NO: 81): D-S/P-R-F-T-Y/F-N-A/Q/P-N-D-PMotif 3b (SEQ ID NO: 82): I-G-N-G-E

The relative positions of motifs 1b and 3b align with residues 231-243and 549-553, respectively, of the 7527 GTF sequence (SEQ ID NO:65) inFIGS. 2A-O. Identification of motifs 1b (SEQ ID NO:81) and 3b (SEQ IDNO:82) in the catalytic domains of GTF enzymes that synthesize lowmolecular weight glucan having 100% alpha-1,3-glycosidic linkagesindicates that each of these unique motifs may be useful for identifyingother GTFs with similarly activity.

Example 6 Production of GTF Enzyme Lacking Sequence Motif 1a

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with a deletion of Motif 1a (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:84), encodingGTF protein 7527-NT-dIS1a (SEQ ID NO:85), was subcloned intopJexpress404® (DNA 2.0, Menlo Park Calif.) to generate the plasmididentified as pMP101. Plasmid pMP101 was used to transform E. coli TOP10cells to generate the strain identified as TOP10/pMP101. It is notedthat a GTF catalytic domain sequence is located at amino acid positions54-941 (approximate) of SEQ ID NO:85.

Production of 7527-NT-dIS1a enzyme (SEQ ID NO:85) with E. coli andproduction of glucan polymer using this enzyme were performed asdescribed above (General Methods). The glucan product is insoluble, andlikely comprises only alpha-glycosidic linkages. The intrinsic viscosityand branching of the glucan product (analyzed as described in GeneralMethods) are listed in Table 5 below.

Example 7 Production of GTF Enzyme Lacking Sequence Motif 2

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with a deletion of Motif 2 (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:86), encodingGTF protein 7527-NT-dIS2 (SEQ ID NO:87), was subcloned intopJexpress404® to generate the plasmid identified as pMP102. PlasmidpMP102 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP102. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-927 (approximate) of SEQID NO:87.

Production of 7527-NT-dIS2 (SEQ ID NO:87) with E. coli and production ofglucan polymer using this enzyme were performed as described above(General Methods). The glucan product is insoluble, and likely comprisesonly alpha-glycosidic linkages. The intrinsic viscosity and branching ofthe glucan product (analyzed as described in General Methods) are listedin Table 5 below.

Example 8 Production of GTF Enzyme Lacking Sequence Motif 3a

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with a deletion of Motif 3a (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:88), encodingGTF protein 7527-NT-dIS3a (SEQ ID NO:89), was subcloned intopJexpress404® to generate the plasmid identified as pMP103. PlasmidpMP103 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP103. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-935 (approximate) of SEQID NO:89.

Production of 7527-NT-dIS3a (SEQ ID NO:89) with E. coli and productionof glucan polymer using this enzyme were performed as described above(General Methods). The glucan product is insoluble, and likely comprisesonly alpha-glycosidic linkages. The intrinsic viscosity and branching ofthe glucan product (analyzed as described in General Methods) are listedin Table 5 below.

Example 9 Production of GTF Enzyme Lacking Sequence Motifs 1a and 2

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with deletion of Motifs 1a and 2 (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:90), encodingGTF protein 7527-NT-dIS1a,2 (SEQ ID NO:91), was subcloned intopJexpress404® to generate the plasmid identified as pMP104. PlasmidpMP104 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP104. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-911 (approximate) of SEQID NO:91.

Production of 7527-NT-dIS1a,2 (SEQ ID NO:91) with E. coli and productionof glucan polymer using this enzyme were performed as described above(General Methods). The glucan product is insoluble, and likely comprisesonly alpha-glycosidic linkages. The intrinsic viscosity and branching ofthe glucan product (analyzed as described in General Methods) are listedin Table 5 below.

Example 10 Production of GTF Enzyme Lacking Sequence Motifs 1a and 3a

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with deletion of Motifs 1a and 3a (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:92), encodingGTF protein 7527-NT-dIS1a,3a (SEQ ID NO:93), was subcloned intopJexpress404® to generate the plasmid identified as pMP105. PlasmidpMP105 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP105. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-919 (approximate) of SEQID NO:93.

Production of 7527-NT-dIS1a,3a (SEQ ID NO:93) with E. coli andproduction of glucan polymer using this enzyme were performed asdescribed above (General Methods). The intrinsic viscosity and branchingof the glucan product (analyzed as described in General Methods) arelisted in Table 5 below.

Example 11 Production of GTF Enzyme Lacking Sequence Motifs 2 and 3a

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with deletion of Motifs 2 and 3a (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:94), encodingGTF protein 7527-NT-dIS2,3a (SEQ ID NO:95), was subcloned intopJexpress404® to generate the plasmid identified as pMP106. PlasmidpMP106 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP106. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-905 (approximate) of SEQID NO:95.

Production of 7527-NT-dIS2,3a (SEQ ID NO:95) with E. coli and productionof glucan polymer using this enzyme were performed as described above(General Methods). The intrinsic viscosity and branching of the glucanproduct (analyzed as described in General Methods) are listed in Table 5below.

Example 12 Production of GTF Enzyme Lacking Sequence Motifs 1a, 2 and 3a

A nucleotide sequence encoding a polypeptide similar to the 7527 GTF ofSEQ ID NO:65, but with deletion of Motifs 1a, 2 and 3a (Example 4), wassynthesized using codons optimized for expression in E. coli (DNA 2.0,Menlo Park Calif.). The nucleic acid product (SEQ ID NO:96), encodingGTF protein 7527-NT-dIS1a,2,3a (SEQ ID NO:97), was subcloned intopJexpress404® to generate the plasmid identified as pMP107. PlasmidpMP107 was used to transform E. coli TOP10 cells to generate the strainidentified as TOP10/pMP107. It is noted that a GTF catalytic domainsequence is located at amino acid positions 54-889 (approximate) of SEQID NO:97.

Production of 7527-NT-dIS1a,2,3a (SEQ ID NO:97) with E. coli andproduction of glucan polymer using this enzyme were performed asdescribed above (General Methods). The intrinsic viscosity and branchingof the glucan product (analyzed as described in General Methods) arelisted in Table 5 below.

Example 13 Analysis of Intrinsic Viscosity and Branching of GlucanProducts Synthesized by GTF Enzymes

This Example describes measuring the intrinsic viscosity (IV) andbranching (g′) of glucan polymer synthesized by each of thedeletion-containing GTF enzymes prepared in Examples 6-12. Thesemeasurements were compared to those obtained with glucan polymerproduced by the 7527 GTF of SEQ ID NO:65, which does not have anyinternal deletions of Motifs 1a, 2 and/or 3a.

It is noted that the glucan polymer synthesized by 7527 GTF, polyalpha-1,3-glucan, has 100% alpha-1,3 linkages and is thus linear (seeTable 4, for example).

The intrinsic viscosity and branching of glucan polymer samples producedby deletion-containing versions of 7527 GTF were analyzed as describedin the General Methods, and are shown in Table 5 below. Glucan polymerproduced by non-deleted 7527 GTF (control), which is listed as “7527-NT”in Table 5, was also analyzed.

TABLE 5 Intrinsic Viscosity (IV) and Branching Index (g′) of GlucanPolymer Produced by Various GTF Enzymes Glucan Product SEQ MissingMeasurement Enzyme ID ID NO Motif(s) IV g′ 7527-NT 65 N/A 206 1.0007527-NT-dlS1a 85 1a 94 0.410 7527-NT-dlS2 87 2 33 0.231 7527-NT-dlS3a 893a 28 0.268 7527-NT-dlS1a, 2 91 1a and 2 21 0.261 7527-NT-dlS1a, 3a 931a and 3a 18 0.215 7527-NT-dlS2, 3a 95 2 and 3a 19 0.256 7527-NT-dlS1a,2, 3a 97 1a, 2 and 3a 22 0.242

As shown in Table 5, glucan produced by each GTF enzyme missing at leastone of Motifs 1a (motif i), 2 (motif ii), or 3a (motif iii) haddecreased intrinsic viscosity (IV) and branching index (g′), as comparedto glucan produced by the corresponding control GTF (7527-NT) havingeach of these motifs. Since reductions in either IV and/or g′ indicateincreased polymer branching, these results demonstrate that each ofMotifs 1a, 2 and 3a may be essential for certain GTF enzymes—ones thatnaturally contain each of these motifs—to produce linearalpha-1,3-glucan polymer.

This observation was not expected, given that some GTF enzymes thatproduce linear product do not contain any of Motifs 1a, 2, or 3 a. Forexample, each of GTFs 5926, 0427, 0874, and 1724 produce polyalpha-1,3-glucan with 100% alpha-1,3 linkages (which is linear) (Table4), despite not having any of these motifs. Indeed, since there appearedto be a correlation between the presence of Motifs 1a, 2 and 3a withincreased glucan product molecular weight (see Example 4), it might havebeen more reasonable to have expected that Motif 1a, 2, and/or 3aremoval would reduce glucan product molecular weight (instead of havingan effect on branching).

Thus, GTF amino acid Motifs 1a, 2 and 3a play a role in production oflinear poly alpha-1,3-glucan by those GTF enzymes that contain thesemotifs

Example 14 GTF Catalytic Domain Activity

This Example describes testing catalytic domain sequences of certainGTFs for the ability to produce insoluble poly alpha-1,3-glucan.Specifically, catalytic domain sequences of GTFs 7527 (SEQ ID NO:65) and5926 (SEQ ID NO:14) were tested for activity.

A GTF catalytic domain sequence having amino acid residues 54-957 of SEQID NO:65 was prepared using the heterologous expression techniquesdescribed above. Briefly, a DNA sequence (codon-optimized for expressionin E. coli) encoding a methionine at the first amino acid positionfollowed by amino acid residues 54-957 of SEQ ID NO:65 was prepared andused to express this catalytic domain sequence. This protein, comparedto the amino acid sequence identified in GENBANK under GI number 47527(SEQ ID NO:60), is truncated by 230 amino acids at the N-terminus and384 amino acids at the C-terminus.

A GTF catalytic domain sequence having amino acid residues 57-906 of SEQID NO:14 was prepared using the heterologous expression techniquesdescribed above. Briefly, a DNA sequence (codon-optimized for expressionin E. coli) encoding a methionine at the first amino acid positionfollowed by amino acid residues 57-906 of SEQ ID NO:14 was prepared andused to express this catalytic domain sequence. This protein, comparedto the amino acid sequence identified in GENBANK under GI number167735926 (SEQ ID NO:83), is truncated by 199 amino acids at theN-terminus and 417 amino acids at the C-terminus.

The above procedures were followed to prepare reaction solutionscontaining either of these GTF catalytic domain sequences. The reactionswere performed at 25° C. and the alpha-1,3-glucan produced in eachreaction was analyzed for DP_(w). The results are provided in Table 6.

TABLE 6 Alpha-1,3-Glucan Polymer Produced by Gtf Enzyme CatalyticDomains Catalytic Initial Domain sucrose % Sucrose Sequence DP_(w) (g/L)consumption 5926 108 150 100 7527 495 142 94

As shown in Table 6, catalytic domain sequences of GTF 7527 (residues54-957 of SEQ ID NO:65) and GTF 5926 (residues 57-906 of SEQ ID NO:14)were able to catalyze production of poly alpha-1,3-glucan. The molecularweight of the poly alpha-1,3-glucan produced by each of these catalyticdomain sequences generally corresponded with the molecular weight of theproduct produced by their counterparts containing both the catalyticdomain and glucan binding domain (refer to activity of SEQ ID NOs:65 and14 in Table 4, DP_(w)150).

Thus, the catalytic domain of a glucosyltransferase enzyme can be usedto produce insoluble poly alpha-1,3-glucan in a reaction solution.

What is claimed is:
 1. A glucosyltransferase enzyme comprising an aminoacid sequence that is at least 97% identical to SEQ ID NO:87, SEQ IDNO:91, SEQ ID NO:95, or SEQ ID NO:97, wherein said glucosyltransferaseenzyme lacks the amino acid sequence of SEQ ID NO:79, and wherein saidglucosyltransferase enzyme produces a branched alpha-glucan polymer. 2.A reaction solution comprising water, sucrose, and a glucosyltransferaseenzyme according to claim
 1. 3. A method for producing a branchedalpha-glucan polymer comprising: (a) contacting at least water, sucrose,and a glucosyltransferase enzyme according to claim 1, whereby branchedalpha-glucan polymer is produced, and b) optionally, isolating thebranched alpha-glucan polymer produced in step (a).
 4. Theglucosyltransferase of claim 1, wherein the glucosyltransferasecomprises an amino acid sequence that is at least 98% identical to SEQID NO:87, SEQ ID NO:91, SEQ ID NO:95, or SEQ ID NO:97.